Lung Volume Reduction Therapy Using Crosslinked Biopolymers

ABSTRACT

One aspect of the present invention relates to bronchoscopic lung volume reduction using solutions of biopolymers that can be polymerized in situ with a crosslinker and a polymeric additive which accelerates the cross-linking reaction. In certain embodiments, the biopolymer solutions can be in the form of a foam or gel. The biopolymer compositions disclosed herein may also be used for indications other than lung volume reduction, such as sealing fistulas or performing emergency tamponade of vessels.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/909,806, filed Apr. 3, 2007.

BACKGROUND

Emphysema is a common form of chronic obstructive pulmonary disease(COPD) that affects between 1.5 and 2 million Americans, and 3 to 4times that number of patients worldwide. [American Thoracic SocietyConsensus Committee “Standards for the diagnosis and care of patientswith chronic obstructive pulmonary disease,” Am. J. Resp. Crit. CareMed. 1995, 152, 78-83; and Pauwels, R., et al. “Global strategy for thediagnosis, management, and prevention of chronic obstructive pulmonarydisease,” Am. J. Resp. Crit. Care Med. 2001, 163, 1256-1271.] It ischaracterized by destruction of the small airways and lung parenchymadue to the release of enzymes from inflammatory cells in response toinhaled toxins. [Stockley, R. “Neutrophils and protease/antiproteaseimbalance,” Am. J. Resp. Crit. Care Med. 1999, 160, S49-S52.] Althoughthis inflammatory process is usually initiated by cigarette smoking,once emphysema reaches an advanced stage, it tends to progress in anunrelenting fashion, even in the absence of continued smoking [Rutgers,S. R., et al. “Ongoing airway inflammation inpatients with COPD who donot currently smoke,” Thorax 2000, 55, 12-18.]

The class of enzymes that are responsible for producing tissue damage inemphysema are known as proteases. These enzymes are synthesized byinflammatory cells within the body and when released, they act todegrade the collagen and elastin fibers which provide mechanicalintegrity and elasticity to the lung. [Jeffery, P. “Structural andinflammatory changes in COPD: a comparison with asthma,” Thorax 1998,53, 129-136.] The structural changes that result from the action ofthese enzymes are irreversible, cumulative, and are associated with lossof lung function that eventually leaves patients with limitedrespiratory reserve and reduced functional capacity. [Spencer, S. et al.“Health status deterioration inpatients with chronic obstructivepulmonary disease,” Am. J. Resp. Crit. Care Med. 2001, 163, 122-128; andMoy, M. L., et al. “Health-related quality of life improves followingpulmonary rehabilitation and lung volume reduction surgery,” Chest 1999,115, 383-389.]

In contrast to other common forms of COPD, such as asthma and chronicbronchitis for which effective medical treatments exist, conventionalmedical treatment is of limited value in patients with emphysema.Although emphysema, asthma, and chronic bronchitis each cause chronicairflow obstruction, limit exercise capacity, and cause shortness ofbreath, the site and nature of the abnormalities in asthma and chronicbronchitis are fundamentally different from those of emphysema. Inasthma and chronic bronchitis, airflow limitation is caused by airwaynarrowing due to smooth muscle constriction and mucus hyper-secretion.Pharmacologic agents that relax airway smooth muscle and loosenaccumulated secretions are effective at improving breathing function andrelieving symptoms. Agents that act in this way include beta-agonist andanti-cholinergic inhalers, oral theophylline preparations, leukotrieneantagonists, steroids, and mucolytic drugs.

In contrast, airflow limitation in emphysema is not primarily due toairway narrowing or obstruction, but due to loss of elastic recoilpressure as a consequence of tissue destruction. Loss of recoil pressurecompromises the ability to fully exhale, and leads to hyper-inflationand gas trapping. Although bronchodilators, anti-inflammatory agents,and mucolytic agents are frequently prescribed for patients withemphysema, they are generally of limited utility since they are intendedprimarily for obstruction caused by airway disease; these classes ofcompounds do nothing to address the loss of elastic recoil that isprincipally responsible for airflow limitation in emphysema. [Barnes, P.“Chronic Obstructive Pulmonary Disease,” N. Engl. J. Med. 2000, 343(4),269-280.]

While pharmacologic treatments for advanced emphysema have beendisappointing, a non-medical treatment of emphysema has recentlyemerged, which has demonstrated clinical efficacy. This treatment islung volume reduction surgery (LVRS). [Flaherty, K. R. and F J. Martinez“Lung volume reduction surgery for emphysema,” Clin. Chest Med. 2000,21(4), 819-48.]

LVRS was originally proposed in the late 1950s by Dr. Otto Brantigan asa surgical remedy for emphysema. The concept arose from clinicalobservations which suggested that in emphysema the lung was “too large”for the rigid chest cavity, and that resection of lung tissuerepresented the best method of treatment since it would reduce lungsize, allowing it to fit and function better within the chest. Initialexperiences with LVRS confirmed that many patients benefitedsymptomatically and functionally from the procedure. Unfortunately,failure to provide objective outcome measures of improvement, coupledwith a 16% operative mortality, led to the initial abandonment of LVRS.

LVRS was accepted for general clinical application in 1994 through theefforts of Dr. Joel Cooper, who applied more stringent pre-operativeevaluation criteria and modern post-operative management schemes toemphysema patients. [Cooper, J. D., et al. “Bilateral pneumonectomy forchronic obstructive pulmonary disease,” J. Thorax. Cardiovascular.Surge. 1995, 109, 106-119.] Cooper reported dramatic improvements inlung function and exercise capacity in a cohort of 20 patients withadvanced emphysema who had undergone LVRS. There were no deaths at90-day follow-up, and physiological and functional improvements weremarkedly better than had been achieved with medical therapy alone.

While less dramatic benefits have been reported by most other centers,LVRS has nevertheless proven to be effective for improving respiratoryfunction and exercise capacity, relieving disabling symptoms of dispend,and improving quality of life in patients with advanced emphysema.[Gelb, A. F., et al. “Mechanism of short-term improvement in lungfunction after emphysema resection,” Am. J. Respir. Crit. Care Med.1996, 154, 945-51; Gelb, A. F., et al. “Serial lung function and elasticrecoil 2 years after lung volume reduction surgery for emphysema,” Chest1998, 113(6), 1497-506; Criner, G. and G. E. D'Alonzo, Jr., “Lung volumereduction surgery: finding its role in the treatment of patients withsevere COPD,” J. Am. Osteopath. Assoc. 1998, 98(7), 371; Brenner, M., etal. “Lung volume reduction surgery for emphysema,” Chest 1996, 110(1),205-18; and Ingenito, E. P., et al. “Relationship between preoperativeinspiratory lung resistance and the outcome of lung-volume-reductionsurgery for emphysema,” N. Engl. J. Med. 1998, 338, 1181-1185.] Thebenefits of volume reduction have been confirmed in numerous cohortstudies, several recently-completed small randomized clinical trials,and the National Emphysema Treatment Trial (NETT). [Goodnight-White, S.,et al. “Prospective randomized controlled trial comparing bilateralvolume reduction surgery to medical therapy alone inpatients with severeemphysema,” Chest 2000, 118(Suppl 4), 1028; Geddes, D., et al.“L-effects of lung volume reduction surgery inpatients with emphysema,”N. Eng. J. Med. 2000, 343, 239-245; Pompeo, E., et al. “Reductionpneumoplasty versus respiratory rehabilitation in severe emphysema: arandomized study,” Ann. Thorac. Surg. 2000, 2000(70), 948-954; andFishman, A., et al. “A randomized trial comparing lung-volume-reductionsurgery with medical therapy for severe emphysema,” N. Eng. J. Med.2003, 348(21): 2059-73.] On average, 75-80% of patients have experienceda beneficial clinical response to LVRS (generally defined as a 12% orgreater improvement in FEV, at 3 month follow-up). The peak responsesgenerally occur at between 3 and 6 months post-operatively, andimprovement has lasted several years. [Cooper, J. D. and S. S. Lefrak“Lung-reduction surgery: 5 years on,” Lancet 1999, 353(Suppl 1), 26-27;and Gelb, A. F., et al. “Lung function 4 years after lung volumereduction surgery for emphysema,” Chest 1999, 116(6), 1608-15.] Resultsfrom NETT have further shown that in a subset of patients withemphysema, specifically those with upper lobe disease and reducedexercise capacity, mortality at 29 months is reduced.

Collectively, these data indicate that LVRS improves quality of life andexercise capacity in many patients, and reduces mortality in a smallerfraction of patients, with advanced emphysema. Unfortunately, NETT alsodemonstrated that the procedure is very expensive when considered interms of Quality Adjusted Life Year outcomes, and confirmed that LVRS isassociated with a 5-6% 90 day mortality. [Chatila, W., S. Furukawa, andG. J. Criner, “Acute respiratory failure after lung volume reductionsurgery,” Am. J. Respir. Crit. Care Med. 2000, 162, 1292-6; Cordova, F.C. and G. J. Criner, “Surgery for chronic obstructive pulmonary disease:the place for lung volume reduction and transplantation,” Curr. Opin.Pulm. Med. 2001, 7(2), 93-104; Swanson, S. J., et al. “No-cutthoracoscopic lung placation: a new technique for lung volume reductionsurgery,” J. Am. Coll. Surg. 1997, 185(1), 25-32; Sema, D. L., et al.“Survival after unilateral versus bilateral lung volume reductionsurgery for emphysema,” J. Thorac. Cardiovasc. Surg. 1999, 118(6),1101-9; and Fishman, A., et al. “A randomized trial comparinglung-volume-reduction surgery with medical therapy for severeemphysema,” N. Engl. J. Med. 2003, 348(21), 2059-73.] In addition,morbidity following LVRS is common (40-50%) and includes a highincidence of prolonged post-operative air-leaks, respiratory failure,pneumonia, cardiac arrhythmias, and gastrointestinal complications. Lessinvasive and less expensive alternatives that could produce the samephysiological effect are desirable.

A hydrogel-based system for achieving lung volume reduction has beendeveloped and tested, and its effectiveness confirmed in both healthysheep, and sheep with experimental emphysema. [Ingenito, E. P., et al.“Bronchoscopic Lung Volume Reduction Using Tissue EngineeringPrinciples,” Am. J. Respir. Crit. Care Med. 2003, 167, 771-778.] Thissystem uses a rapidly-polymerizing, fibrin-based hydrogel that can bedelivered through a dual lumen catheter into the lung using abronchoscope. The fibrin-based system effectively blocks collateralventilation, inhibits surfactant function to promote collapse, andinitiates a remodeling process that proceeds over a 4-6 week period.Treatment results in consistent, effective lung volume reduction. Thesestudies have confirmed the safety and effectiveness of usingfibrin-based hydrogels in the lung to achieve volume reduction therapy.

SUMMARY

One aspect of the invention relates to bronchoscopic lung volumereduction using a composition comprising a crosslinker, a biopolymerthat can be polymerized in situ with the crosslinker, and a polymericadditive, which accelerates the cross-linking reaction. In certainembodiments, the composition comprising a crosslinker, polymericadditive, and biopolymer is in the form of a gel or foam. In certainembodiments, the biopolymer contains a plurality of free amino groups.In certain embodiments, the biopolymer is a protein, polysaccharide orpolynucleotide. In certain embodiments, the biopolymer is a protein. Incertain embodiments, the biopolymer is a protein selected from the groupconsisting of actin, albumin, alpha-globulin, beta-globulin,gamma-globulin, cadherin, calmodulin, calbindin, casein, catenin,collagens, C-reactive protein, cholesterylester transfer protein,cytokines, DNA binding proteins, dystrophin, elastin, ferritin, fetuin,fibrinogen, fibrin, fibroin, fibronectin, gelatin, hemoglobin, histones,insulin, epidermal growth factor, heparin, interleukins, insulin-likegrowth factor, integrin, keratin, kinases, laminin, lysozyme, myoglobin,myosin, reelin, rhodopsin, selectin, transthyretin, thrombin, tubulin,trypsin, utrophin, and vinculin. In certain embodiments, the biopolymeris albumin. Another aspect of the invention relates to bronchoscopiclung volume reduction using gels or foams generated from solutions ofbiopolymers which contain free amine groups that can be polymerized insitu with di-, tri, or poly-aldehydes. Yet another aspect of theinvention relates to a method of bronchoscopic lung volume reductionusing a cross-linked gel or foam generated from a solution of an albuminprotein, which contains free amine groups, that can be polymerized insitu via an aldehyde-containing cross-linker; and a polymeric additivewhich accelerates the cross-linking reaction. In certain embodiments,the albumin protein is a mammalian serum albumin. In certainembodiments, the albumin protein is bovine serum albumin or human serumalbumin. In certain embodiments, the aldehyde-containing cross-linker isa dialdehyde. In certain embodiments, the aldehyde-containingcross-linker is glutaraldehyde. In certain embodiments, the compositionsand methods cause minimal toxicity, are injectable through a catheter,and polymerize rapidly enough to prevent solution from spilling backinto the airways following injection.

In certain embodiments, the above-mentioned method for reducing lungvolume in a patient comprises the steps of administering to a region ofthe lungs of a patient a composition comprising a biopolymer, across-linker, and a polymeric additive; wherein said polymeric additiveaccelerates a cross-linking reaction between the biopolymer and thecross-linker. In certain embodiments, the composition is administeredusing a bronchoscope or catheter. In certain embodiments, theabove-mentioned method for reducing lung volume in a patient furthercomprises the step of advancing into a region of a patient's lung viasaid patient's trachea a catheter lumen through a bronchoscope. Incertain embodiments, the composition is a foam or gel. In certainembodiments, the composition is a foam. In certain embodiments, thecomposition is a gel. In certain embodiments, the composition furthercomprises a gas. In certain embodiments, the above-mentioned method forreducing lung volume in a patient comprises the steps of advancing intoa region of a patient's lung via said patient's trachea a catheter lumenthrough a bronchoscope; and administering, through the catheter, a gelor foam composition comprising an albumin protein, a cross-linker, and apolymeric additive; wherein said polymeric additive accelerates across-linking reaction between the albumin protein and the cross-linker.In certain embodiments the gel or foam composition is formed bycombining the albumin protein, the cross-linker, and the polymericadditive and then foaming the mixture with a gas. Because of therelatively slow polymerization in some embodiments, there is time tofoam the solution prior to administration to the patient. Alternatively,the gel or foam composition is formed by combining the albumin proteinand the polymeric additive and then foaming the mixture; to this mixtureis added the cross-linker. In both cases, a foam composition isadministered to the patient (e.g., via a single lumen catheter), andresults in a cross-linked foam in the patient's lung. In certainembodiments, the compositions and methods described herein are intendedfor use in the treatment of patients with emphysema.

It should be appreciated that compositions of the invention also mayinclude one or more additional compounds (e.g., therapeutic compound(s),stabilizing compound(s), antibiotic(s), growth factor(s), etc.),buffers, salts, surfactants, anti-surfactants, lipids, excipients,and/or other suitable compounds. In certain embodiments, compositions ofthe invention may be sterilized.

In certain embodiments, compositions of the invention may be used topromote one or more of the following responses when contacted to atissue in a body: sclerosis (hardening of tissue), fibrosis (excessfibrous connective tissue), wound healing, tissue sealing, localmicrovascular thrombosis (blood clot), cellular necrosis or apoptosis(cell death), tumor regression, cell lysis, or any combination thereof.

Additional advantages and novel features of the present invention willbecome apparent from the following detailed description of variousnon-limiting embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a graph showing the effects of albumin and glutaraldehyde(GA) concentration on polymerization time at room temperature. Each dataset corresponds to a different albumin concentration from 20 to 36%.

FIG. 2 depicts a graph showing the effects of albumin/glutaraldehyderatio (Alb/GA) on polymerization time at room temperature. Each data setcorresponds to a different glutaraldehyde concentration from 0.3 to 2%.

FIG. 3 depicts two graphs showing the effects of adding 5, 10 or 15% [A]polyvinylpyrrolidone (PVP) or [B] dextran to a mixture of 25% albuminand 0.25% glutaraldehyde (GA) at room temperature.

FIG. 4 depicts a bar graph comparing the results depicted in FIG. 3.

FIG. 5 depicts a graph showing the effects of glutaraldehyde (GA)concentration on polymerization time at room temperature. Each data setcorresponds to a different polyvinylpyrrolidone (PVP) concentration from0 to 9%.

FIG. 6 depicts a bar graph comparing the effects of adding 4.5 or 9%polyvinylpyrrolidone (PVP) to a mixture of 22.5% human serum albumin(HSA) and 0.3% glutaraldehyde (GA) at room temperature.

FIG. 7 depicts Table 1, showing the foamability of a mixture of 22.5%human serum albumin with 4.5% polyvinylpyrolidine; Table 2, showing theinjectability of a the same human serum albumin and glutaraldehydemixture; and Table 3, showing treatment groups and results for variousbovine serum albumin and glutaraldehyde mixtures.

FIG. 8 depicts Table 4, showing treatment results obtained with amixture of 25% BSA, 10% PVP and 0.25% GA; Table 5, showing treatmentgroups corresponding to administration of the same mixture; and Table 6,showing gross necropsy findings corresponding to administration of thesame mixture.

FIG. 9 depicts a graph showing results of lung volume reduction study insheep using a mixture of 25% BSA, 6% PVP and 0.25% GA.

DETAILED DESCRIPTION

One aspect of the invention relates to compositions and methods fortreatment of patients with advanced emphysema. In certain embodiments,the invention relates to a system for achieving lung volume reductiontherapy, wherein a composition is injected into the lung. In certainembodiments, the composition is a foam or gel. In certain embodiments,the composition is a foam. In certain embodiments, the composition is agel. In certain embodiments, the invention relates to a system forachieving lung volume reduction therapy, wherein a composition isinjected into the lung as a gas-containing foam. Delivery of theinventive compositions may follow an initial pretreatment designed toprime the treatment area by causing collapse and/or by removing thesurface lining cells of the treatment area. However, certain inventivecompositions can, by themselves, provide this function and do notrequire pretreatment.

The compositions in various forms (e.g., foam or gel) serve several keyfunctions that are beneficial for promoting lung volume reduction:blocking collateral ventilation by coating the interstices of the lungsurface, a step that prevents rapid re-inflation of the treatment area;ensuring that reagents remain localized to the treatment area, sinceupon polymerization, the foam or gel becomes trapped in the smallairways and alveoli of the lung, preventing flow beyond the intendedtreatment site; and filling the treatment area, displacing air andforming a bridge between adjacent regions of lung tissue.

In certain embodiments, to be effective as a volume reducing agent inthe lung, the precursors of the cross-linked foam or gel compositionmust have sufficiently fast polymerization kinetics and physicalproperties to allow for endoscopic delivery. The compositions must havefavorable biocompatibility profiles, show rapid polymerization, and havemechanical properties such that following polymerization the firmness ofthe foam or gel composition does not mechanically injure adjacent softlung tissues. Further, the compositions must have initial viscositiesthat will allow them to be injected through a small-bore catheter. Inaddition, the foam or gel composition must have acceptablepharmacokinetic degradation profiles in vivo. The inventive compositionsdescribed herein which posses some or all of these features should besatisfactory for achieving bronchoscopic lung volume reduction therapy.

Herein are described cross-linked biopolymer (e.g., albumin) albuminfoams or gels that possess some or all of these properties. In addition,in certain embodiments, the cross-linked albumin foams or gels of theinvention may show superior properties to some known LVRT compositionsbecause of improved tissue adhesion; the foams or gels of the inventionmay have minimal seepage and may be self-healing (i.e., substantiallyless cracks or breaks might be formed in the solidified mass).

It is known that albumin proteins and glutaraldehyde can be combined toform a rapidly polymerizing, biocompatible tissue glue: Bioglue® is acommercially available albumin/glutaraldehyde tissue glue which contains36 w % bovine serum albumin and 2 w % GA (final concentrations aftermixing). However, in order for such a glue to be efficacious forbronchoscopic lung volume reduction (BLVR), it must have specificproperties, including: a polymerization time long enough to allowdelivery to the lung via a bronchoscopically placed catheter (greaterthan about 1 min); fluid mechanical properties that allow injectionthrough a bronchoscopically-guided small bore catheter; andpolymerization time short enough to allow practical procedure lengthwithout spillage from the treatment site (less than about 5 minutes).Because of the rapid rate of polymerization of Bioglue® (less thanthirty seconds; data not shown), the Bioglue® composition is not wellsuited for LVRT. Moreover, decreasing the ratio of bovine serum albuminto glutaraldehyde in order to achieve the desired polymerizationkinetics results in a glutaraldehyde concentration which is highlytoxic. Glutaraldehyde toxicity is well known. See, for example, Speer,D. P. et al. J. Biomedical Mat. Res. 1980, 14, 753-764; Huang-Lee, L. L.H. et al. J. Biomedical Mat. Res. 1990, 24, 1185-1201; Furst, W. et al.Ann. Thorac. Surg. 2005, 79, 1522-1529; and Zeiger, E. et al. MutationRes. 2005, 589, 136-151.

Remarkably, as disclosed herein, the use of a polymeric additive enablescompositions that foam or gel with the desired characteristics. Thereare many advantages to the compositions and methods described herein. Insome respects, the compositions described herein are chemically simplerthat current LVRT compositions. In certain embodiments, the chemicalsare less expensive. In certain embodiments, the foams and gels of theinvention have better space filling characteristics than fibrin-basedhydrogel systems, meaning that smaller amounts of material can be usedto collapse larger lung volumes. In addition, in certain embodiments,there appears to be less potential for systemic toxicity than with someother LVRT approaches.

DEFINITIONS

For convenience, certain terms employed in the specification andappended claims are collected here. These definitions should be read inlight of the entire disclosure and understood as by a person of skill inthe art.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The term “biodegradable” is intended to mean any component capable ofdisappearing by progressive degradation (metabolism).

The term “contrast-enhancing” refers to materials capable of beingmonitored during injection into a mammalian subject by methods formonitoring and detecting such materials, for example by radiography orfluoroscopy. An example of a contrast-enhancing agent is a radiopaquematerial. Contrast-enhancing agents including radiopaque materials maybe either water soluble or water insoluble. Examples of water solubleradiopaque materials include metrizamide, iopamidol, iothalamate sodium,iodomide sodium, and meglumine. Examples of water insoluble radiopaquematerials include metals and metal oxides such as gold, titanium,silver, stainless steel, oxides thereof, aluminum oxide, zirconiumoxide, etc.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₁-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to aboutten carbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The term “aralkyl” is art-recognized and refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, naphthalene, anthracene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring may be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, fluoroalkyl, cyano,or the like. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings (the rings are “fused rings”) wherein at leastone of the rings is aromatic, e.g., the other cyclic rings may becycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles.

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “heterocyclyl”, “heteroaryl”, or “heterocyclic group” areart-recognized and refer to 3- to about 10-membered ring structures,alternatively 3- to about 7-membered rings, whose ring structuresinclude one to four heteroatoms. Heterocycles may also be polycycles.Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidone, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, fluoroalkyl, cyano,or the like.

The terms “polycyclyl” or “polycyclic group” are art-recognized andrefer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, fluoroalkyl, cyano,or the like.

The term “carbocyclyl” or “carbocycle” is art-recognized and refers toan aromatic or non-aromatic ring in which each atom of the ring iscarbon.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to SH; the term “hydroxyl”means —OH; and the term “sulfonyl” is art-recognized and refers to SO₂⁻. “Halide” designates the corresponding anion of the halogens, and“pseudohalide” has the definition set forth in “Advanced InorganicChemistry” by Cotton and Wilkinson.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines.

The term “amido” is art recognized as an amino-substituted carbonyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g., alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

Aliphatic is a C₁-C₁₂ chain, wherein one or more carbon atoms isoptionally substituted with a heteroatom selected from the groupconsisting of oxygen, nitrogen or sulfur. Each carbon is optionallysubstituted with a functional group selected from the group consistingof hydroxyl, thiol, amino, alkyl, alkoxy, thioalkyl, amionalkyl, aryl,aryloxy, thioaryl, arylamino, heteroaryl and cycloalkyl. Aliphatic alsoincludes optionally substituted C₁-C₁₂ alkenyl and alkynyl groups.Straight-chain or branched C₁-C₁₂-alkyl group is selected from the groupconsisting of methyl, ethyl, propyl, isopropyl, n-butyl, 2-butyl,sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 2-methylbutyl, 3-methylbutyl,1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl,1-ethylpropyl, n-hexyl, 2-hexyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl,1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl,2-ethylbutyl, 1-ethyl-2-methylpropyl, n-heptyl, 2-heptyl, 3-heptyl,2-ethylpentyl, 1-propylbutyl, n-octyl, 2-ethylhexyl, 2-propylheptyl,nonyl and decyl.

Cycloaliphatic is a C₃-C₇ cycloalkyl selected from the group consistingof cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. TheC₃-C₇ cycloalkyl is optionally substituted with 1, 2, 3, 4 or 5substituents selected from the group consisting of hydroxyl, thiol,amino, alkyl, alkoxy, thioalkyl, amionalkyl, aryl, aryloxy, thioaryl,arylamino, heteroaryl and cycloalkyl.

Aromatic is an aryl group selected from the group consisting of phenyl,tolyl, xylyl, mesityl, naphthyl, fluorenyl, anthracenyl, phenanthrenyl,pyridyl, and naphthacenyl, wherein the aryl group is optionallysubstituted with 1, 2, 3, 4 or 5 substituents selected from the groupconsisting of alkyl, alkoxy, thioalkyl, amino, nitro, trifluoromethyl,aryl, halo and cyano. Aromatic dialdehydes include isophthalaldehyde,phthalaldehyde and terephthalaldehyde.

Heterocycloaliphatic is a C₄-C₇ ring optionally substituted with 1, 2 or3 heteroatoms selected from the group consisting of oxygen, nitrogen andsulfur. Each carbon is optionally substituted with a functional groupselected from the group consisting of hydroxyl, thiol, amino, alkyl,alkoxy, thioalkyl, amionalkyl, aryl, aryloxy, thioaryl, arylamino,heteroaryl and cycloalkyl. Heterocycloaliphatic group includespyrrolidinyl, piperidinyl, 2,2,6,6-tetramethylpiperidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, morpholidinyl,thiazolidinyl, isothiazolidinyl, isoxazolidinyl, piperazinyl,tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl and dioxanyl.

Heterocyclic is a heterocycloaromatic selected from the group consistingof pyridyl, quinolinyl, acridinyl, pyridazinyl, pyrimidinyl andpyrazinyl, wherein the heterocycloaromatic is optionally substitutedwith 1, 2 or 3 substituents selected from the group consisting of alkyl,alkoxy, thioalkyl, amino, nitro, trifluoromethyl, aryl, halo and cyano.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the present invention mayexist in particular geometric or stereoisomeric forms. The presentinvention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

Biopolymers

The biopolymers for use in the present invention can be proteins,polysaccharides or polynucleotides, wherein the monomer units are aminoacids, saccharides or nucleic acids, respectively. Examples ofbiopolymers include, but are not limited to actin, albumin,alpha-globulin, beta-globulin, gamma-globulin, cadherin, calmodulin,calbindin, casein, catenin, celluloses, chitin, collagens, C-reactiveprotein, cholesterylester transfer protein, chondroitin sulfate,cytokines, DNA, DNA binding proteins, dystrophin, elastin, ferritin,fetuin, fibrinogen, fibrin, fibroin, fibronectin, gelatin, hemoglobin,histones, insulin, epidermal growth factor, heparin, interleukins,insulin-like growth factor, integrin, keratan sulfate, keratin, kinases,laminin, lysozyme, myoglobin, myosin, reelin, rhodopsin, RNA, selectin,transthyretin, thrombin, tubulin, trypsin, utrophin and vinculin.

In certain embodiments, the biopolymer of the present invention is apoly(amino acid), comprising

at least 90% of amino acids selected from the group consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, methionine,phenylalanine, serine, threonine, tyrosine and valine; and

no more than 10% of amino acids selected from the group consisting oflysine and tryptophan.

In certain embodiments, the biopolymer of the present invention is apoly(aminosaccharide) of formula I or II:

wherein independently for each occurrence:m is 1 or 2;

R is OH or NH₂; and

the stereochemical configuration at a stereocenter is R or S.

Albumins

Albumin refers generally to any protein with water solubility, which ismoderately soluble in concentrated salt solutions, and which experiencesheat coagulation (protein denaturation). Substances containing albumin,such as egg white, are called albuminoids. The most well-known type ofalbumin is serum albumin in the blood, but there is also the storageprotein ovalbumin in egg white, and other storage albumins in the seedsof some plants.

Serum albumin is the most abundant blood plasma protein and is producedin the liver and forms a large proportion of all plasma protein. Humanserum albumin, a water-soluble protein of 585 amino acids with amolecular weight of 66 kD, is the most abundant protein in plasma(3.5-5.0 g/100 mL in blood plasma), but also exists in lowerconcentrations in extra vascular fluids. It has a large number ofcharged amino acids (about 100 negative charges and 100 positivecharges) with an isoelectric point of 5.0 and a net negative charge of−15 at a plasma pH of 7.4, and attracts both anions and cations. Incertain embodiments, the albumin protein of the invention is a mammalianserum albumin, human serum albumin, porcine serum albumin and/or bovineserum albumin. In certain embodiments, the albumin protein of theinvention is a human serum albumin and/or bovine serum albumin. Incertain embodiments, the albumin protein of the invention is a humanserum albumin. In certain embodiments, the albumin is a recombinantprotein.

Cross-Linkers

One embodiment of the present invention relates to the cross-linking ofbiopolymers (e.g., albumin proteins). It is well known in the art thatbifunctional “cross-linking” reagents contain two reactive groups, thusproviding a means of covalently linking two target groups. When thebiopolymer to be cross-linked comprises nucleophilic moieties, thereactive groups in a chemical cross-linking reagent typically belong tothe classes of electrophilic functional groups, e.g., succinimidylesters, maleimides, idoacetamides, and aldehydes. However, when thebiopolymer to be cross-linked comprises electrophilic moieties, thereactive groups in a chemical cross-linker may be nucleophilicfunctional groups, e.g., alcohols, thiols and amines. Bifunctionalcross-linking reagents can be divided in homobifunctional,heterobifunctional and zero-length bifunctional cross-linking reagents.In homobifunctional cross-linking reagents, the reactive groups areidentical. In heterobifunctional cross-linking reagents, the reactivegroups are not identical. The “zero-length” cross-linking reagent formsa chemical bond between two groups utilizing a single functional group(e.g., a carbonyl moiety derived from carbonyl diimidazole) or withoutitself being incorporated into the product. For example, a water-solublecarbodiimide (EDAC) may be used to couple carboxylic acids to amines. Inaddition to the traditional bifunctional cross-linking reagents, anoncovalent interaction between two molecules that has very slowdissociation kinetics can also function as a crosslink. For example,reactive derivatives of phospholipids can be used to link the liposomesor cell membranes to antibodies or enzymes. Biotinylation andhaptenylation reagents can also be thought of as heterobifunctionalcross-linking reagents because they comprise a chemically reactive groupas well as a biotin or hapten moiety that binds with high affinity toavidin or an anti-hapten antibody, respectively.

In certain embodiments, the cross-linkers of the present invention arehomobifunctional cross-linkers (e.g., dialdehydes). In otherembodiments, the cross-linkers of the present invention arehomopolyfunctional cross-linking reagents.

In certain embodiments, the cross-linkers of the present invention aredi- or polyaldehydes. As will be appreciated by one skilled in the art,aldehydes described herein can exist as hydrates in aqueous solution,e.g., existing as hemi-acetals in aqueous solution. In certainembodiments, such hydrates can revert back to the corresponding aldehydeand/or ketone for cross-linking. In some embodiments, hydrates ofaldehydes and/or hydrates of other cross-linking activating moieties arethemselves capable of bringing about cross-linking

In certain embodiments, the cross-linker of the invention is W—X_(n)—W,wherein independently for each occurrence,

W is

X is —C(R)₂—; or, for at most two, three, four, five, six, seven oreight occurrences, X is —C(═O)—, —O—, —S—, —N(R)—, —N(C(O)R)—,—C(R)═C(R)—, —C≡C—, —C≡N—, —C(R)═N—, a cycloalkyl diradical, aheterocycloalkyl diradical, an aryl diradical, or a heteroaryldiradical;

Y is a bond, —C(R)₂—, —C(═O)—, —O—, —S—, —N(R)—, or —N(C(O)R)—;

R is hydrogen, alkyl, lower alkyl, carbocyclyl, alkenyl, lower alkenyl,alkynyl, lower alkynyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and

n is 1-20 inclusive.

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein W is

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein W is

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein W is

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein X is —CR₂—. In certain embodiments,the present invention relates to the aforementioned cross-linker,wherein X is —CH₂—.

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein Y is a bond, —CR₂—, or —O—. Incertain embodiments, the present invention relates to the aforementionedcross-linker, wherein Y is a bond, or —CH₂—.

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein R is hydrogen or lower alkyl. Incertain embodiments, the present invention relates to the aforementionedcross-linker, wherein R is hydrogen.

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein n is 1-10 inclusive. In certainembodiments, the present invention relates to the aforementionedcross-linker, wherein n is 2. In certain embodiments, the presentinvention relates to the aforementioned cross-linker, wherein n is 3. Incertain embodiments, the present invention relates to the aforementionedcross-linker, wherein n is 4. In certain embodiments, the presentinvention relates to the aforementioned cross-linker, wherein n is 5. Incertain embodiments, the present invention relates to the aforementionedcross-linker, wherein n is 6.

In certain embodiments, the cross-linker is glutaraldehyde. As mentionedabove, it has been found that the absolute local concentration ofglutaraldehyde must be maintained at or below a level that does notproduce undesired excessive local toxicity. At concentrations of 0.75%or greater, glutaraldehyde produces significant tissue necrosis.Concentrations below this level produce limited local toxicityassociated with clinically acceptable side effects. In otherembodiments, other di- and polyaldehydes, such as glyoxal may be used.

In certain embodiments, the crosslinker of the present invention isrepresented by the following formula:

wherein independently for each occurrence

n is 0-12;

m is 0-12; and

X is a di-radical of an aliphatic, cycloaliphatic, aromatic,heterocycloaliphatic or heterocyclic moiety.

In certain embodiments, the crosslinker of the invention is representedby the following formula:

wherein independently for each occurrence

n is 0-12;

m is 0-12; and

R and R₁ are each independently hydrogen, aliphatic, cycloaliphatic,aromatic, heterocycloaliphatic or heterocyclic moiety.

In certain embodiments, the cross-linker is of biological origin. Incertain embodiments, the cross-linker is an aldehyde. In certainembodiments, said aldehyde is an oxidized polysaccharide. In certainembodiments, the aldehyde is an oxidized polysaccharide, thepolysaccharide being at least one from the group of dextran, chitin,starch, agar, cellulose, alginic acid, glycosaminoglycans, hyaluronicacid, chondroitin sulfate and derivatives thereof. In certainembodiments, the aldehdye is dextranaldehyde. The aldehyde, especiallythe dextranaldehyde, preferably has a molecular weight of about 60,000to 600,000, in particular about 200,000. Higher molecular weights, inparticular of at least 200,000, may result in high degrees ofcrosslinking

In certain embodiments, the aldehyde cross-linker is advantageouslypartially or completely masked. The purpose of the masking, especiallyof oxidized polyaldehydes, is to prevent the formation of intermolecularacetals and thus ensure the stability of the solutions.

In certain embodiments, a method of the invention results in overalllung volume reduction of about 0.5% to about 40%. In certainembodiments, a method of the invention results in overall lung volumereduction of about 0.5% to about 30%. In certain embodiments, a methodof the invention results in overall lung volume reduction of about 0.5%to about 20%. In certain embodiments, a method of the invention resultsin overall lung volume reduction of about 0.5% to about 10%. Suchreduction may be achieved upon a single or multiple administrations ofcompositions of the present invention.

Ratio of Biopolymer to Cross-Linker

As mentioned above, one of the drawbacks to some known cross-linkedalbumin compositions is that the high concentration of cross-linkerneeded to achieve workable polymerization kinetics leads to toxicity.One aspect of the present invention relates to compositions wherein theratio of the biopolymer to the cross-linker is greater than about 20:1.In certain embodiments, the ratio of the biopolymer to the cross-linkeris greater than about 30:1; about 40:1; about 50:1; about 60:1; about70:1; about 80:1; about 90:1; about 100:1; about 110:1; about 120:1;about 130:1; about 140:1; about 150:1; about 160:1; about 170:1; about180:1; about 190:1; or about 200:1. All ratios are weight ratios; inother words, a ratio of about 20:1 means the weight of the biopolymer isabout twenty times the weight of the cross-linker.

Cross-Linking Rate Modifiers

In certain embodiments, a polymeric additive may be used as a ratemodifier to modify the rate of the cross-linking reaction. In certainembodiments, the polymeric additives may accelerate the formation of aSchiff base between amines in the biopolymers and aldehydes in thecross-linker. While not intending to be bound by any mechanistic theory,the effect of the polymeric additives may be to sequester water and thusincrease the effective concentration of cross-linker. Alternatively, orin addition, the polymeric additives may catalytically, perhaps throughhydrogen bonding, accelerate the cross-linking reaction.

In certain embodiments, the polymeric additive is based on polymers ofvinylpyrrolidone. The structure of vinylpyrrolidone is

In certain embodiments, the polymeric additive ispoly(vinylpyrrolidone). In other embodiments, the polymer of theinvention is a copolymer of vinyl pyrrolidone. For example, polymericadditives of the invention include vinylpyrrolidone/dimethylaminoethylmethacrylate copolymer, vinylpyrrolidone/styrene copolymer, andvinylpyrrolidone/vinyl acetate copolymer. In certain copolymers there isabout 25 mol % vinylpyrrolidone; about 40 mol % vinylpyrrolidone; about50 mol % vinylpyrrolidone; about 60 mol % vinylpyrrolidone; about 75 mol% vinylpyrrolidone; about 85 mol % vinylpyrrolidone; or about 95 mol %vinylpyrrolidone.

In certain embodiments, the polymeric additive is based on dextran.Dextran is a complex, branched polysaccharide made of many glucosemolecules joined into chains of varying lengths. The straight chainconsists of α1→6 glycosidic linkages between glucose molecules, whilebranches begin from α1→3 linkages (and in some cases, α1→2 and α1→4linkages as well). Dextran is synthesized from sucrose by Leuconostocmesenteroides streptococcus and Streptococcus mutans and is alsoproduced by bacteria and yeast.

In certain embodiments, the polymeric additive is based on ethyleneglycol polymers. Polyethylene glycol (PEG) and polyethylene oxide (PEO)are polymers composed of repeating subunits of identical structure,called monomers, and are the most commercially important polyethers.Poly(ethylene glycol) or poly(ethylene oxide) refers to an oligomer orpolymer of ethylene oxide. The two names are chemically synonymous, buthistorically PEG has tended to refer to shorter polymers, PEO to longer.As used herein, polyethylene glycol refers to both PEG and PEO. PEG andPEO are liquids or low-melting solids, depending on their molecularweights. Both are prepared by polymerization of ethylene oxide. WhilePEG and PEO with different molecular weights find use in differentapplications and have different physical properties (e.g., viscosity)due to chain length effects, their chemical properties are nearlyidentical. Derivatives of PEG and PEO are in common use, the most commonderivative being the methyl ether (methoxypoly(ethylene glycol)),abbreviated mPEG. Their melting points vary depending on the formulaweight of the polymer. PEG or PEO has the following structure:HO(CH₂CH₂O)_(r)H. The numbers that are often included in the names ofPEGs and PEOs indicate their average molecular weights, e.g., a PEG withr equal to about 80 would have an average molecular weight ofapproximately 3500 Daltons and would be labeled PEG 3500.

In certain embodiments the polymeric additive is based on acrylic acidpolymers. Carbomer is a non-proprietary name for these materials. Theyare high molecular weight polymers prepared by cross-linking acrylicacids with the likes of allyl ether pentaerythritol, allyl ether ofsucrose, or allyl ether of propylene. Such polymers also go by the namesAcritamer® or Carbopol®. The chemical name and CAS registry number forthe class is carboxypolymethylene [54182-57-9]. Exemplary carbomers arecarbomer 910 [91315-32-1], carbomer 934 [9007-16-3], carbomer 934P[9003-01-4] and carbomer 940 [76050-42-5]. These polymers containbetween 56-68% of carboxylic acid groups, calculated on a dry basis. Ablend of two or more carbomers of differing molecular weight may be usedto modify and manipulate the polymerization rate.

Many of the polymers above are produced as mixtures of molecules with adistribution of molecular weights, i.e., they are polydisperse. The sizedistribution can be characterized statistically by its weight averagemolecular weight (M_(w)) and its number average molecular weight(M_(n)), the ratio of which is called the polydispersity index(M_(w)/M_(n)). M_(w) and M_(n) can be measured by mass spectroscopy.

In certain embodiments, the cross-linking reaction substantially occursin less than or equal to about 1 minute, less than or equal to about 90seconds, less than or equal to about 2 minutes, less than or equal toabout 150 seconds, less than or equal to about 3 minutes, less than orequal to about 4 minutes, less than or equal to about 5 minutes, lessthan or equal to about 6 minutes, less than or equal to about 10minutes.

Compositions

In certain embodiments, the present invention relates to compositionswherein the biopolymer is about 10-40% by weight of said composition. Incertain embodiments, the biopolymer is about 15-35% by weight of saidcompositions. In certain embodiments, the biopolymer is about 20-30% byweight of said composition. In certain embodiments, the biopolymer isabout 25% by weight of said composition. In certain embodiments, thebiopolymer is about 22.5% by weight of said composition.

In certain embodiments, the present invention relates to compositionswherein the crosslinker is about 0.1-2% by weight of said composition.In certain embodiments, the crosslinker is about 0.1-1% by weight ofsaid composition. In certain embodiments, the crosslinker is about0.1-0.5% by weight of said composition. In certain embodiments, thecrosslinker is about 0.3% by weight of said composition. In certainembodiments, the crosslinker is about 0.25% by weight of saidcomposition.

In certain embodiments, the present invention relates to compositionswherein the polymeric additive is about 0-15% by weight of saidcomposition. In certain embodiments, the polymeric additive is about2-12% by weight of said composition. In certain embodiments, thepolymeric additive is about 10% by weight of said composition. Incertain embodiments, the polymeric additive is about 4.5% by weight ofthe composition.

In certain embodiments, the present invention relates to compositionswherein the biopolymer is about 10-40% by weight of said composition;the crosslinker is about 0.1-2% by weight of said composition; and thepolymeric additive is about 0-15% by weight of said composition. Incertain embodiments, the biopolymer is about 25% by weight of saidcomposition; the crosslinker is about 0.25% by weight of saidcomposition; and the polymeric additive is about 10% by weight of saidcomposition. In certain embodiments, the biopolymer is about 22.5% byweight of said composition, the crosslinker is about 0.3% by weight ofsaid composition; and the polymeric additive is about 4.5% by weight ofthe composition.

In certain embodiments, the present invention relates to compositions,wherein bovine serum albumin is about 25% by weight of said composition,GA is about 0.25% by weight of the composition; and PVP is about 10% byweight of said compositions. In certain embodiments, human serum albuminis about 22.5% by weight of said composition, GA is about 0.3% by weightof said composition; and PVP is about 4.5% by weight of saidcomposition.

Foaming Modifiers

In certain embodiments, a foaming modifier facilitates the generation ofa stable foam. In other words, in certain embodiments a foaming modifiermay be introduced into an inventive composition to facilitate theformation of a foamed composition. Examples of such a foaming modifierinclude tissue compatible surfactants, tyloxapol, poloxamers,poloxamines, phospholipids, and glycerol. Illustrative of these foamingmodifiers are non-toxic surfactants including, but are not limited to,fats or proteins in edible foams. However, the surfactant may be anionic or non-ionic surfactant depending on the intended application. Theionic surfactants including, for example, anionic surfactants, such assodium stearate, sodium dodecyl sulfate, α-olefinsulfonate andsulfoalkylamides and cationic surfactants, such asalkyldimethylbenzylammonium salts, alkyltrimethylammonium salts andalkylpyridinium salts; and amphoteric surfactants such as imidazolinesurfactants. The non-ionic surfactants include, for example,polyethylene oxide alkyl ethers, polyethylene oxide alkylphenyl ethers,glycerol fatty acid esters, sorbitan fatty acid esters, sucrose fattyacid esters, and the like.

Additional surfactants which may be used include surfactants such asTriton x-100, beractant, colfosceril, and/or palmitate; anionicsurfactants such as sodium tetradecyl sulfate; cationic surfactants suchas tetrabutylammonium bromide and/or butyrylcholine chloride; nonionicsurfactants such as polysorbate 20 (e.g., Tween 20), polysorbate 80(e.g., Tween 80), and/or poloxamers; amphoteric and/or zwitterionicsurfactants such as dodecyldimethyl(3-sulfopropyl)ammonium hydroxide,inner salt; amines, imines and/or amides, such as arginine, imidazole,povidine, tryptamine, and/or urea; alcohols such as ascorbic acid,ethylene glycol, methyl gallate, tannins and/or tannic acid; phosphines,phosphites and phosphonium salts, such as triphenylphosphine and/ortriethyl phosphite; inorganic bases and/or salts, such as calciumsulfate, magnesium hydroxide, sodium silicate, and/or sodium bisulfite;sulfur compounds such as polysulfides and/or thiourea; polymeric cyclicethers such as calixarenes, crown ethers, monensin, nonactin, and/orpolymeric epoxides; cyclic and acyclic carbonates; organometallics(e.g., naphthenate and manganese acetylacetonate); phase transfercatalysts (e.g., Aliquat 336); and radical initiators and radicals(e.g., di-t-butyl peroxide and/or azobisisobutyronitrile).

Therapeutic Agents

Any of a vast number of therapeutic agents may be incorporated in thefoams or gels used in the methods of the present invention. In general,therapeutic agents which may be incorporated include, withoutlimitation: antiinfectives such as antibiotics and antiviral agents (asmentioned above); analgesics and analgesic combinations; anorexics;antihelmintics; antiarthritics; antiasthmatic agents; anticonvulsants;antidepressants; antidiuretic agents; antidiarrheals; antihistamines;antiinflammatory agents; antimigraine preparations; antinauseants;antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics;antipyretics, antispasmodics; anticholinergics; sympathomimetics;xanthine derivatives; cardiovascular preparations including calciumchannel blockers and beta-blockers such as pindolol and antiarrhythmics;antihypertensives; diuretics; vasodilators including general coronary,peripheral and cerebral; central nervous system stimulants; cough andcold preparations, including decongestants; hormones such as estradioland other steroids, including corticosteroids; hypnotics;immunosuppressives; muscle relaxants; parasympatholytics;psychostimulants; sedatives; and tranquilizers; and naturally derived orgenetically engineered proteins, polysaccharides, glycoproteins, orlipoproteins. Suitable pharmaceuticals for parenteral administration arewell known as is exemplified by the Handbook on Injectable Drugs, 6^(th)Edition, by Lawrence A. Trissel, American Society of HospitalPharmacists, Bethesda, Md., 1990 (hereby incorporated by reference).

The pharmaceutically active compound may be any substance havingbiological activity, including proteins, polypeptides, polynucleotides,nucleoproteins, polysaccharides, glycoproteins, lipoproteins, andsynthetic and biologically engineered analogs thereof. The term“protein” is art-recognized and for purposes of this invention alsoencompasses peptides. The proteins or peptides may be any biologicallyactive protein or peptide, naturally occurring or synthetic.

Examples of proteins include antibodies, enzymes, growth hormone andgrowth hormone-releasing hormone, gonadotropin-releasing hormone, andits agonist and antagonist analogues, somatostatin and its analogues,gonadotropins such as luteinizing hormone and follicle-stimulatinghormone, peptide T, thyrocalcitonin, parathyroid hormone, glucagon,vasopressin, oxytocin, angiotensin I and II, bradykinin, kallidin,adrenocorticotropic hormone, thyroid stimulating hormone, insulin,glucagon and the numerous analogues and congeners of the foregoingmolecules. The pharmaceutical agents may be selected from insulin,antigens selected from the group consisting of MMR (mumps, measles andrubella) vaccine, typhoid vaccine, hepatitis A vaccine, hepatitis Bvaccine, herpes simplex virus, bacterial toxoids, cholera toxinB-subunit, influenza vaccine virus, bordetela pertussis virus, vacciniavirus, adenovirus, canary pox, polio vaccine virus, plasmodiumfalciparum, bacillus calmette geurin (BCG), klebsiella pneumoniae, HIVenvelop glycoproteins and cytokins and other agents selected from thegroup consisting of bovine somatropine (sometimes referred to as BST),estrogens, androgens, insulin growth factors (sometimes referred to asIGF), interleukin I, interleukin II and cytokines Three such cytokinesare interferon-β, interferon-γ and tuftsin.

Examples of bacterial toxoids that may be incorporated in the foams orgels used in the methods of the invention are tetanus, diphtheria,pseudomonas A, mycobaeterium tuberculosis. Examples of that may beincorporated in the compositions used in the occlusion methods of theinvention are HIV envelope glycoproteins, e.g., gp 120 or gp 160, forAIDS vaccines. Examples of anti-ulcer H2 receptor antagonists that maybe included are ranitidine, cimetidine and famotidine, and otheranti-ulcer drugs are omparazide, cesupride and misoprostol. An exampleof a hypoglycaemic agent is glizipide.

Classes of pharmaceutically active compounds which can be loaded intothat may be incorporated in the foams or gels used in the methods of theinvention include, but are not limited to, anti-AIDS substances,anti-cancer substances, antibiotics, immunosuppressants (e.g.,cyclosporine) anti-viral substances, enzyme inhibitors, neurotoxins,opioids, hypnotics, antihistamines, lubricants tranquilizers,anti-convulsants, muscle relaxants and anti-Parkinson substances,anti-spasmodics and muscle contractants, miotics and anti-cholinergics,anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds,anti-hypertensives, analgesics, anti-pyretics and anti-inflammatoryagents such as NSAIDs, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents, specific targeting agents, neurotransmitters, proteins, cellresponse modifiers, and vaccines.

Exemplary pharmaceutical agents considered to be particularly suitablefor incorporation in the foams or gels used in the methods of theinvention include but are not limited to imidazoles, such as miconazole,econazole, terconazole, saperconazole, itraconazole, metronidazole,fluconazole, ketoconazole, and clotrimazole,luteinizing-hormone-releasing hormone (LHRH) and its analogues,nonoxynol-9, a GnRH agonist or antagonist, natural or syntheticprogestrin, such as selected progesterone, 17-hydroxyprogeteronederivatives such as medroxyprogesterone acetate, and 19-nortestosteroneanalogues such as norethindrone, natural or synthetic estrogens,conjugated estrogens, estradiol, estropipate, and ethinyl estradiol,bisphosphonates including etidronate, alendronate, tiludronate,resedronate, clodronate, and pamidronate, calcitonin, parathyroidhormones, carbonic anhydrase inhibitor such as felbamate anddorzolamide, a mast cell stabilizer such as xesterbergsterol-A,lodoxamine, and cromolyn, a prostaglandin inhibitor such as diclofenacand ketorolac, a steroid such as prednisolone, dexamethasone,fluromethylone, rimexolone, and lotepednol, an antihistamine such asantazoline, pheniramine, and histiminase, pilocarpine nitrate, abeta-blocker such as levobunolol and timolol maleate. As will beunderstood by those skilled in the art, two or more pharmaceuticalagents may be combined for specific effects. The necessary amounts ofactive ingredient can be determined by simple experimentation.

By way of example only, any of a number of antibiotics andantimicrobials may be included in the foams or gels used in the methodsof the invention. Antimicrobial drugs preferred for inclusion incompositions used in the methods of the invention include salts oflactam drugs, quinolone drugs, ciprofloxacin, norfloxacin, tetracycline,erythromycin, amikacin, triclosan, doxycycline, capreomycin,chlorhexidine, chlortetracycline, oxytetracycline, clindamycin,ethambutol, hexamidine isethionate, metronidazole, pentamidine,gentamicin, kanamycin, lineomycin, methacycline, methenamine,minocycline, neomycin, netilmicin, paromomycin, streptomycin,tobramycin, miconazole and amanfadine and the like.

By way of example only, in the case of anti-inflammation, non-steroidalanti-inflammatory agents (NSAIDS) may be incorporated in the foams orgels used in the methods of the invention, such as propionic acidderivatives, acetic acid, fenamic acid derivatives, biphenylcarboxylicacid derivatives, oxicams, including but not limited to aspirin,acetaminophen, ibuprofen, naproxen, benoxaprofen, flurbiprofen,fenbufen, ketoprofen, indoprofen, pirprofen, carporfen, and bucloxicacid and the like.

Selected Compositions of the Invention

One aspect of the invention relates to a composition comprising abiopolymer, a cross-linker, and a polymeric additive; wherein thepolymeric additive accelerates a cross-linking reaction between thebiopolymer and the cross-linker

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition is a gel or foam.

In certain embodiments, the present invention relates to theaforementioned composition wherein said biopolymer contains a pluralityof amine groups.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said biopolymer is a protein,polysaccharide, or polynucleotide.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said biopolymer is a protein.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said biopolymer is a proteinselected from the group consisting of actin, albumin, alpha-globulin,beta-globulin, gamma-globulin, cadherin, calmodulin, calbindin, casein,catenin, collagens, C-reactive protein, cholesterylester transferprotein, cytokines, DNA binding proteins, dystrophin, elastin, ferritin,fetuin, fibrinogen, fibrin, fibroin, fibronectin, gelatin, hemoglobin,histones, insulin, epidermal growth factor, heparin, interleukins,insulin-like growth factor, integrin, keratin, kinases, laminin,lysozyme, myoglobin, myosin, reelin, rhodopsin, selectin, transthyretin,thrombin, tubulin, trypsin, utrophin and vinculin.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said biopolymer is albumin.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said albumin is a mammalian albumin.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said albumin is a mammalian serumalbumin.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said albumin is human serum albuminor bovine serum albumin.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said albumin is human serum albumin.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said albumin is bovine serumalbumin.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said albumin is not bovine serumalbumin.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is a dialdehyde ora polyaldehyde.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is a dialdehyde.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is W—X_(n)—W,wherein independently for each occurrence,

W is

X is —C(R)₂—; or, for at most two, three, four, five, six, seven oreight occurrences, X is —C(═O)—, —O—, —S—, —N(R)—, —N(C(O)R)—,—C(R)═C(R)—, —C≡C—, —C≡N—, —C(R)═N—, a cycloalkyl diradical, aheterocycloalkyl diradical, an aryl diradical, or a heteroaryldiradical;

Y is a bond, —C(R)₂—, —C(═O)—, —O—, —S—, —N(R)—, or —N(C(O)R)—;

R is hydrogen, alkyl, lower alkyl, carbocyclyl, alkenyl, lower alkenyl,alkynyl, lower alkynyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; andn is 1-20 inclusive.

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein W is

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein W is

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein W is

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein X is —CR₂—. In certain embodiments,the present invention relates to the aforementioned cross-linker,wherein X is —CH₂—.

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein Y is a bond, —CR₂—, or —O—. Incertain embodiments, the present invention relates to the aforementionedcross-linker, wherein Y is a bond, or —CH₂—.

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein R is hydrogen or lower alkyl. Incertain embodiments, the present invention relates to the aforementionedcross-linker, wherein R is hydrogen.

In certain embodiments, the present invention relates to theaforementioned cross-linker, wherein n is 1-10 inclusive. In certainembodiments, the present invention relates to the aforementionedcross-linker, wherein n is 2. In certain embodiments, the presentinvention relates to the aforementioned cross-linker, wherein n is 3. Incertain embodiments, the present invention relates to the aforementionedcross-linker, wherein n is 4. In certain embodiments, the presentinvention relates to the aforementioned cross-linker, wherein n is 5. Incertain embodiments, the present invention relates to the aforementionedcross-linker, wherein n is 6.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is notglutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said crosslinker is represented bythe following formula:

wherein independently for each occurrence n is 0-12;

-   -   m is 0-12; and

X is a di-radical of an aliphatic, cycloaliphatic, aromatic,heterocycloaliphatic or heterocyclic moiety.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said crosslinker of the invention isrepresented by the following formula:

wherein independently for each occurrence n is 0-12;

m is 0-12; and

R and R₁ are each independently hydrogen, aliphatic, cycloaliphatic,aromatic, heterocycloaliphatic or heterocyclic moiety.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is water solubleat a concentration of at least about 1 mg/mL.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is water solubleat a concentration of at least about 2.5 mg/mL.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is water solubleat a concentration of at least about 5 mg/mL.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is water solubleat a concentration of at least about 10 mg/mL.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said cross-linker is water solubleat a concentration of at least about 20 mg/mL.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said biopolymer is a mammalian serumalbumin; and the cross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said biopolymer is human serumalbumin or bovine serum albumin; and the cross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned composition, wherein the weight ratio of biopolymer tocross-linker is about 30:1 to about 200:1.

In certain embodiments, the present invention relates to theaforementioned composition, wherein the weight ratio of biopolymer tocross-linker is about 60:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned composition, wherein the weight ratio of biopolymer tocross-linker is about 80:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned composition, wherein the weight ratio of biopolymer tocross-linker is about 100:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned composition, wherein the weight ratio of biopolymer tocross-linker is about 80:1 to about 100:1.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said polymeric additive acceleratesSchiff base formation.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said polymeric additive is apoly(vinylpyrrolidone) polymer or copolymer, dextran, a poly(ethyleneglycol) or a carbomer.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said polymeric additive is apoly(vinylpyrrolidone) or dextran.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition further comprises agas.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said gas is non-toxic.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said gas is oxygen or air.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said polymeric additive has a weightaverage molecular weight of between about 25,000 g/mol and about 250,000g/mol.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said polymeric additive has a weightaverage molecular weight of between about 25,000 g/mol and about 150,000g/mol.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said polymeric additive has a weightaverage molecular weight of about 50,000 g/mol.

In certain embodiments, the present invention relates to theaforementioned composition, further comprising a foam-modifying agent.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said foam-modifying agent is asurfactant.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said foam-modifying agent istyloxapol, a poloxamer, a poloxamine, a phospholipid, or glycerol.

In certain embodiments, the present invention relates to theaforementioned composition, further comprising an anti-infective.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said anti-infective is selected fromthe group consisting of an aminoglycoside, a tetracycline, asulfonamide, p-aminobenzoic acid, a diaminopyrimidine, a quinolone, aβ-lactam, a β-lactamase inhibitor, chloraphenicol, a macrolide,penicillins, cephalosporins, linomycin, clindamycin, spectinomycin,polymyxin B, colistin, vancomycin, bacitracin, isoniazid, rifampin,ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin,a sulfone, clofazimine, thalidomide, a polyene antifungal, flucytosine,imidazole, triazole, griseofulvin, terconazole, butoconazole ciclopirax,ciclopirox olamine, haloprogin, tolnaftate, naftifine, and terbinafine,or a combination thereof

In certain embodiments, the present invention relates to theaforementioned composition, wherein said anti-infective is tetracycline.

In certain embodiments, the present invention relates to theaforementioned composition, further comprising a contrast-enhancingagent.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said contrast-enhancing agent isselected from the group consisting of radiopaque materials, paramagneticmaterials, heavy atoms, transition metals, lanthanides, actinides, dyes,and radionuclide-containing materials.

In certain embodiments, the present invention relates to theaforementioned composition, wherein upon combination of said biopolymer;said cross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about10 minutes.

In certain embodiments, the present invention relates to theaforementioned composition, wherein upon combination of said biopolymer;said cross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about 7minutes.

In certain embodiments, the present invention relates to theaforementioned composition, wherein upon combination of said biopolymer;said cross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about 5minutes.

In certain embodiments, the present invention relates to theaforementioned composition, wherein upon combination of said biopolymer;said cross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 2 minutes to about6 minutes.

In certain embodiments, the present invention relates to theaforementioned composition, wherein upon combination of said biopolymer;said cross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 3 minutes to about5 minutes.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition substantiallydegrades under physiological conditions in about 1 to about 12 weeks.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition substantiallydegrades under physiological conditions in about 1 to about 6 weeks.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition substantiallydegrades under physiological conditions in about 1 to about 4 weeks.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition substantiallydegrades under physiological conditions in about 2 to about 5 weeks.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition is in contact witha mammalian tissue.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition is in contact witha mammalian pulmonary tissue.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition contacts aninterior surface of a mammalian pulmonary tissue.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition contacts theinterior surface of mammalian alveoli.

In certain embodiments, the present invention relates to theaforementioned composition, wherein said composition contacts theinterior surface of mammalian alveoli and partially or completely fillsthe mammalian alveoli.

Selected Methods of the Invention

Aspects of the invention relate to certain formulations of compositionsthat are useful for non-surgical lung volume reduction. According to theinvention, lung volume reduction, a procedure that reduces lung size byremoving damaged (e.g., over-expanded) regions of the lung, can beaccomplished non-surgically by procedures carried out through thepatient's trachea (e.g., by inserting devices and substances through abronchoscope), rather than by procedures that disrupt the integrity ofthe chest wall [Ingenito et al., Am. J. Resp. Crit. Care Med. 2001, 164,295-301; Ingenito et al., Am. J. Resp. Crit. Care Med. 2000, 161, A750;and Ingenito et al., Am. J. Resp. Crit. Care Med. 2001, 163, A957.] Inone aspect of the invention, non-surgical lung volume reduction isperformed by introducing a material (e.g., a biopolymer foam or gel)into a target region of the lung to promote collapse of the targetregion. In one embodiment, the material promotes stable collapse byadhering to the collapsed tissue together and/or by promoting scarringof the collapsed tissue.

Once a patient is determined to be a candidate for LVR, the targetregion of the lung can be identified using radiological studies (e.g.,chest X-rays) and computed tomography scans. When the LVR procedure issubsequently performed, the patient is anesthetized and intubated, andcan be placed on an absorbable gas (e.g., at least 90% oxygen and up to100% oxygen) for a specified period of time (e.g., approximately 30minutes). The region(s) of the lung that were first identifiedradiologically are then identified bronchoscopically.

Suitable bronchoscopes include those manufactured by Pentax, Olympus,and Fujinon, which allow for visualization of an illuminated field. Thephysician guides the bronchoscope into the trachea and through thebronchial tree so that the open tip of the bronchoscope is positioned atthe entrance to target region (i.e., to the region of the lung that willbe reduced in volume). The bronchoscope can be guided throughprogressively narrower branches of the bronchial tree to reach varioussubsegments of either lung. For example, the bronchoscope can be guidedto a subsegment within the upper lobe of the patient's left lung.

In certain embodiments, a balloon catheter may be guided through thebronchoscope to a target region of the lung. When the catheter ispositioned within the bronchoscope, the balloon is inflated so thatmaterial passed through the catheter will be contained in regions of thelung distal to the balloon. This is particularly may be useful in themethods of the present invention, which include the introduction ofcompositions of the invention into the selected region of the lung. Theballoon (or balloon-like structure) may be spherical, cylindrical, orany other shape. The distended balloon (or balloon-like structure) mayhave a diameter of at least about 0.1 mm, at least about 0.5 mm, atleast about 1.0 mm, at least about 1.5 mm, at least about 3 mm, at leastabout 4 mm, at least about 5 mm, at least about 6 mm, at least about 7mm, at least about 8 mm, at least about 9 mm, or at least about 10 mm.The balloon diameter may be less than about 30 mm, less than about 20mm, less than about 15 mm, or less than about 12 mm. The diameterselected can help position the balloon (or balloon-like structure) in asegmental bronchi, subsegmental bronchi, bronchiole and/or alveoluswithin a deep region of the lung. This anchoring and/or positioning canfacilitate delivery of a composition of the present invention to theselected localized region of damaged lung tissue.

One aspect of the invention relates to a method for reducing lung volumein a patient by administering, a composition comprising a biopolymer, across-linker, and a polymeric additive; wherein said polymeric additiveaccelerates a cross-linking reaction between the biopolymer and thecross-linker.

In certain embodiments, the method further comprises the step ofadvancing into a region of a patient's lung via said patient's trachea acatheter lumen through a bronchoscope.

In certain embodiments, the composition further comprises a gas.

In certain embodiments, the present invention relates to theaforementioned method, wherein said gas is non-toxic.

In certain embodiments, the present invention relates to theaforementioned method, wherein said gas is oxygen or air.

In certain embodiments, the components of the composition are preferablymixed together shortly before application. This mixing can take placefor example with the aid of a double-barrel syringe in which thecomponents are forced into a joint ejection tube in which a static mixeris present. The components are mixed together by the static mixer in theejection tube and are ejected, before substantial cross-linking hasoccurred, from the syringe onto the catheter.

For example, in certain embodiments, the present invention relates tothe aforementioned method, further comprising the step of preparing afoam composition by using a gas to foam a mixture of a biopolymer, across-linker, and a polymeric additive; wherein the polymeric additiveaccelerates a cross-linking reaction between the biopolymer and thecross-linker.

In other embodiments, the present invention relates to theaforementioned method, further comprising the steps of using a gas tofoam a mixture of a biopolymer, and a polymeric additive, therebyforming a foamed mixture; and then preparing a foam composition byadding a cross-linker to the foamed mixture; wherein the polymericadditive accelerates a cross-linking reaction between the biopolymer andthe cross-linker. In certain embodiments, the present invention relatesto the aforementioned method, wherein said method results in thedeflation and atelectasis of said region of the lung.

In certain embodiments, the present invention relates to theaforementioned method, wherein said region of the lung has little or nophysiological function.

In certain embodiments, the present invention relates to theaforementioned method wherein said biopolymer contains a plurality ofamine groups.

In certain embodiments, the present invention relates to theaforementioned method, wherein said biopolymer is a protein,polysaccharide, or polynucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein said biopolymer is a protein.

In certain embodiments, the present invention relates to theaforementioned method, wherein said biopolymer is a protein selectedfrom the group consisting of actin, albumin, alpha-globulin,beta-globulin, gamma-globulin, cadherin, calmodulin, calbindin, casein,catenin, collagens, C-reactive protein, cholesterylester transferprotein, cytokines, DNA binding proteins, dystrophin, elastin, ferritin,fetuin, fibrinogen, fibrin, fibroin, fibronectin, gelatin, hemoglobin,histones, insulin, epidermal growth factor, heparin, interleukins,insulin-like growth factor, integrin, keratin, kinases, laminin,lysozyme, myoglobin, myosin, reelin, rhodopsin, selectin, transthyretin,thrombin, tubulin, trypsin, utrophin and vinculin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said biopolymer is albumin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said albumin is a mammalian albumin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said albumin is a mammalian serumalbumin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said albumin is human serum albumin orbovine serum albumin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said albumin is human serum albumin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said albumin is bovine serum albumin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said albumin is not bovine serum albumin.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is a dialdehyde or apolyaldehyde.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is a dialdehyde.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is W—X_(n)—W, whereinindependently for each occurrence,

W is

X is —C(R)₂—; or, for at most two, three, four, five, six, seven oreight occurrences, X is —C(═O)—, —O—, —S—, —N(R)—, —N(C(O)R)—,—C(R)═C(R)—, —C≡C—, —C≡N—, —C(R)═N—, a cycloalkyl diradical, aheterocycloalkyl diradical, an aryl diradical, or a heteroaryldiradical;

Y is a bond, —C(R)₂—, —C(═O)—, —O—, —S—, —N(R)—, or —N(C(O)R)—;

R is hydrogen, alkyl, lower alkyl, carbocyclyl, alkenyl, lower alkenyl,alkynyl, lower alkynyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and

n is 1-20 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein W is

In certain embodiments, the present invention relates to theaforementioned method, wherein W is

In certain embodiments, the present invention relates to theaforementioned method, wherein W is

In certain embodiments, the present invention relates to theaforementioned method, wherein X is —CR₂—. In certain embodiments, thepresent invention relates to the aforementioned method, wherein X is—CH₂—.

In certain embodiments, the present invention relates to theaforementioned method, wherein Y is a bond, —CR₂—, or —O—. In certainembodiments, the present invention relates to the aforementioned method,wherein Y is a bond, or —CH₂—.

In certain embodiments, the present invention relates to theaforementioned method, wherein R is hydrogen or lower alkyl. In certainembodiments, the present invention relates to the aforementioned method,wherein R is hydrogen.

In certain embodiments, the present invention relates to theaforementioned method, wherein n is 1-10 inclusive. In certainembodiments, the present invention relates to the aforementioned method,wherein n is 2. In certain embodiments, the present invention relates tothe aforementioned method, wherein n is 3. In certain embodiments, thepresent invention relates to the aforementioned method, wherein n is 4.In certain embodiments, the present invention relates to theaforementioned method, wherein n is 5. In certain embodiments, thepresent invention relates to the aforementioned method, wherein n is 6.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is not glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is represented by thefollowing formula:

wherein independently for each occurrence

n is 0-12;

m is 0-12; and

X is a di-radical of an aliphatic, cycloaliphatic, aromatic,heterocycloaliphatic or heterocyclic moiety.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is represented by thefollowing formula:

wherein independently for each occurrence

n is 0-12;

m is 0-12; and

R and R₁ are each independently hydrogen, aliphatic, cycloaliphatic,aromatic, heterocycloaliphatic or heterocyclic moiety.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is water soluble at aconcentration of at least about 1 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is water soluble at aconcentration of at least about 2.5 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is water soluble at aconcentration of at least about 5 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is water soluble at aconcentration of at least about 10 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein said cross-linker is water soluble at aconcentration of at least about 20 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein said biopolymer is a mammalian serumalbumin; and the cross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned method, wherein said biopolymer is human serum albumin orbovine serum albumin; and the cross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned method, wherein the weight ratio of biopolymer tocross-linker is about 30:1 to about 200:1.

In certain embodiments, the present invention relates to theaforementioned method, wherein the weight ratio of biopolymer tocross-linker is about 60:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned method, wherein the weight ratio of biopolymer tocross-linker is about 80:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned method, wherein the weight ratio of biopolymer tocross-linker is about 100:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned method, wherein the weight ratio of biopolymer tocross-linker is about 80:1 to about 100:1.

In certain embodiments, the present invention relates to theaforementioned method, wherein said polymeric additive acceleratesSchiff base formation.

In certain embodiments, the present invention relates to theaforementioned method, wherein said polymeric additive is apoly(vinylpyrrolidone) polymer or copolymer, dextran, a poly(ethyleneglycol) or a carbomer.

In certain embodiments, the present invention relates to theaforementioned method, wherein said polymeric additive is apoly(vinylpyrrolidone) or dextran.

In certain embodiments, the present invention relates to theaforementioned method, wherein said polymeric additive has a weightaverage molecular weight of between about 25,000 g/mol and about 250,000g/mol.

In certain embodiments, the present invention relates to theaforementioned method, wherein said polymeric additive has a weightaverage molecular weight of between about 25,000 g/mol and about 150,000g/mol.

In certain embodiments, the present invention relates to theaforementioned method, wherein said polymeric additive has a weightaverage molecular weight of about 50,000 g/mol.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition further comprises afoam-modifying agent.

In certain embodiments, the present invention relates to theaforementioned method, wherein said foam-modifying agent is asurfactant.

In certain embodiments, the present invention relates to theaforementioned method, wherein said foam-modifying agent is tyloxapol, apoloxamer, a poloxamine, a phospholipid, or glycerol.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition further comprises ananti-infective.

In certain embodiments, the present invention relates to theaforementioned method, wherein said anti-infective is selected from thegroup consisting of an aminoglycoside, a tetracycline, a sulfonamide,p-aminobenzoic acid, a diaminopyrimidine, a quinolone, a β-lactam, aβ-lactamase inhibitor, chloraphenicol, a macrolide, penicillins,cephalosporins, linomycin, clindamycin, spectinomycin, polymyxin B,colistin, vancomycin, bacitracin, isoniazid, rifampin, ethambutol,ethionamide, aminosalicylic acid, cycloserine, capreomycin, a sulfone,clofazimine, thalidomide, a polyene antifungal, flucytosine, imidazole,triazole, griseofulvin, terconazole, butoconazole ciclopirax, ciclopiroxolamine, haloprogin, tolnaftate, naftifine, and terbinafine, or acombination thereof

In certain embodiments, the present invention relates to theaforementioned method, wherein said anti-infective is tetracycline.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition further comprises acontrast-enhancing agent.

In certain embodiments, the present invention relates to theaforementioned method, wherein said contrast-enhancing agent is selectedfrom the group consisting of radiopaque materials, paramagneticmaterials, heavy atoms, transition metals, lanthanides, actinides, dyes,and radionuclide-containing materials.

In certain embodiments, the present invention relates to theaforementioned method, wherein the total amount of the composition isbetween about 5 mL and about 300 mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein the total amount of the composition isbetween about 10 mL and about 100 mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein the total amount of the composition isbetween about 10 mL and about 50 mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein upon combination of said albumin; saidcross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about10 minutes.

In certain embodiments, the present invention relates to theaforementioned method, wherein upon combination of said albumin; saidcross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about 7minutes.

In certain embodiments, the present invention relates to theaforementioned method, wherein upon combination of said albumin; saidcross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about 5minutes.

In certain embodiments, the present invention relates to theaforementioned method, wherein upon combination of said albumin; saidcross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 2 minutes to about6 minutes.

In certain embodiments, the present invention relates to theaforementioned method, wherein upon combination of said albumin; saidcross-linker; and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 3 minutes to about5 minutes.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition substantially degradesunder physiological conditions in about 1 to about 12 weeks.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition substantially degradesunder physiological conditions in about 1 to about 6 weeks.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition substantially degradesunder physiological conditions in about 1 to about 4 weeks.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition substantially degradesunder physiological conditions in about 2 to about 5 weeks.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a human.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient has emphysema.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient has suffered a traumaticinjury of the lung.

In certain embodiments, the present invention relates to theaforementioned method, further comprising the step of collapsing theregion of the lung.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition is in contact with amammalian tissue.

In certain embodiments, the present invention relates to theaforementioned method, wherein said foam composition is in contact witha mammalian pulmonary tissue.

In certain embodiments, the present invention relates to theaforementioned method, wherein said foam composition contacts aninterior surface of a mammalian pulmonary tissue.

In certain embodiments, the present invention relates to theaforementioned method, wherein said foam composition contacts theinterior surface of mammalian alveoli.

In certain embodiments, the present invention relates to theaforementioned method, wherein said foam composition contacts theinterior surface of mammalian alveoli and partially or completely fillsthe mammalian alveoli.

Selected Kits of the Invention

This invention also provides kits for conveniently and effectivelyimplementing the methods of this invention. Such kits comprise any ofthe biopolymers, polymeric additives, and/or cross-linkers of thepresent invention or a combination thereof, and a means for facilitatingtheir use consistent with methods of this invention. Such kits provide aconvenient and effective means for assuring that the methods arepracticed in an effective manner. The compliance means of such kitsincludes any means which facilitates practicing a method of thisinvention. Such compliance means include instructions, packaging, anddispensing means, and combinations thereof. Kit components may bepackaged for either manual or partially or wholly automated practice ofthe foregoing methods. In other embodiments, this invention contemplatesa kit including albumins and/or cross-linkers of the present invention,and optionally instructions for their use.

Any of these kits can contain devices used in non-surgical lung volumereduction. For example, they can also contain a catheter (e.g., asingle- or multi-lumen (e.g., dual-lumen) catheter that, optionally,includes a balloon or other device suitable for inhibiting airflowwithin the respiratory tract), tubing or other conduits for removingmaterial (e.g., solutions, including those that carry debridedepithelial cells) from the lung, a stent or a valve or other device thatmay be placed in an airway to block or reduce airflow into or out of alung or lung region, and/or a bronchoscope.

One aspect of the invention relates to a kit, comprising: a firstcontainer comprising a first amount of a first mixture comprising abiopolymer; a second container comprising a second amount of a secondmixture comprising a cross-linker; a third container comprising a thirdamount of a third mixture comprising a polymeric additive which modifiesthe rate of cross-linking; and instructions for use in lung volumereduction therapy.

Another aspect of the invention relates to a kit, comprising: a firstcontainer comprising a first amount of a first mixture comprising analbumin; a second container comprising a second amount of a secondmixture comprising a cross-linker; a third container comprising a thirdamount of a third mixture comprising a polymeric additive; andinstructions for use in lung volume reduction therapy.

In certain embodiments, the present invention relates to theaforementioned kit, wherein the total amount of the first mixture,second mixture, and third mixture is between about 5 mL and about 300mL.

In certain embodiments, the present invention relates to theaforementioned kit, wherein the total amount of the first mixture,second mixture, and third mixture is between about 10 mL and about 100mL.

In certain embodiments, the present invention relates to theaforementioned kit, wherein the total amount of the first mixture,second mixture and third mixture is between about 10 mL and about 50 mL.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said biopolymer contains a plurality ofamine groups.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said biopolymer is a protein,polysaccharide, or polynucleotide.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said biopolymer is a protein.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said biopolymer is a protein selected fromthe group consisting of actin, albumin, alpha-globulin, beta-globulin,gamma-globulin, cadherin, calmodulin, calbindin, casein, catenin,collagens, C-reactive protein, cholesterylester transfer protein,cytokines, DNA binding proteins, dystrophin, elastin, ferritin, fetuin,fibrinogen, fibrin, fibroin, fibronectin, gelatin, hemoglobin, histones,insulin, epidermal growth factor, heparin, interleukins, insulin-likegrowth factor, integrin, keratin, kinases, laminin, lysozyme, myoglobin,myosin, reelin, rhodopsin, selectin, transthyretin, thrombin, tubulin,trypsin, utrophin and vinculin.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said protein is albumin.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said albumin is a mammalian albumin.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said albumin is a mammalian serum albumin.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said albumin is human serum albumin orbovine serum albumin.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said albumin is human serum albumin.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said albumin is bovine serum albumin.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said albumin is not bovine serum albumin.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is a dialdehyde or apolyaldehyde.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is a dialdehyde.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is W—X_(n)—W, whereinindependently for each occurrence,

W is

X is —C(R)₂—; or, for at most two, three, four, five, six, seven oreight occurrences, X is —C(═O)—, —O—, —S—, —N(R)—, —N(C(O)R)—,—C(R)═C(R)—, —C≡C—, —C≡N—, —C(R)═N—, a cycloalkyl diradical, aheterocycloalkyl diradical, an aryl diradical, or a heteroaryldiradical;

Y is a bond, —C(R)₂—, —C(═O)—, —O—, —S—, —N(R)—, or —N(C(O)R)—;

R is hydrogen, alkyl, lower alkyl, carbocyclyl, alkenyl, lower alkenyl,alkynyl, lower alkynyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and

n is 1-20 inclusive.

In certain embodiments, the present invention relates to theaforementioned kit, wherein W is

In certain embodiments, the present invention relates to theaforementioned kit, wherein W is

In certain embodiments, the present invention relates to theaforementioned kit, wherein W is

In certain embodiments, the present invention relates to theaforementioned kit, wherein X is —CR₂—. In certain embodiments, thepresent invention relates to the aforementioned kit, wherein X is —CH₂—.

In certain embodiments, the present invention relates to theaforementioned kit, wherein Y is a bond, —CR₂—, or —O—. In certainembodiments, the present invention relates to the aforementioned kit,wherein Y is a bond, or —CH₂—.

In certain embodiments, the present invention relates to theaforementioned kit, wherein R is hydrogen or lower alkyl. In certainembodiments, the present invention relates to the aforementioned kit,wherein R is hydrogen.

In certain embodiments, the present invention relates to theaforementioned kit, wherein n is 1-10 inclusive. In certain embodiments,the present invention relates to the aforementioned kit, wherein n is 2.In certain embodiments, the present invention relates to theaforementioned kit, wherein n is 3. In certain embodiments, the presentinvention relates to the aforementioned kit, wherein n is 4. In certainembodiments, the present invention relates to the aforementioned kit,wherein n is 5. In certain embodiments, the present invention relates tothe aforementioned kit, wherein n is 6.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is not glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said crosslinker is represented by thefollowing formula:

wherein independently for each occurrence

n is 0-12;

m is 0-12; and

X is a di-radical of an aliphatic, cycloaliphatic, aromatic,heterocycloaliphatic or heterocyclic moiety.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said crosslinker is represented by thefollowing formula:

wherein independently for each occurrence

n is 0-12;

m is 0-12; and

R and R₁ are each independently hydrogen, aliphatic, cycloaliphatic,aromatic, heterocycloaliphatic or heterocyclic moiety.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said polymeric additive accelerates Schiffbase formation.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said polymeric additive is apoly(vinylpyrrolidone) polymer or copolymer, dextran, a poly(ethyleneglycol) or a carbomer.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said polymeric additive is apoly(vinylpyrrolidone) or dextran.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is water soluble at aconcentration of at least about 1 mg/mL.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is water soluble at aconcentration of at least about 2.5 mg/mL.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is water soluble at aconcentration of at least about 5 mg/mL.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is water soluble at aconcentration of at least about 10 mg/mL.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said cross-linker is water soluble at aconcentration of at least about 20 mg/mL.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said polymeric additive has a weight averagemolecular weight of between about 25,000 g/mol and about 250,000 g/mol.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said polymeric additive has a weight averagemolecular weight of between about 25,000 g/mol and about 150,000 g/mol.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said polymeric additive has a weight averagemolecular weight of about 50,000 g/mol.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said biopolymer is albumin; said polymericadditive is poly(vinylpyrrolidone) or dextran; and the cross-linker isglutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said albumin is a mammalian serum albumin;said polymeric additive is poly(vinylpyrrolidone) or dextran; and thecross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said albumin is human serum albumin orbovine serum albumin; said polymeric additive is poly(vinylpyrrolidone)or dextran; and the cross-linker is glutaraldehyde.

In certain embodiments, the present invention relates to theaforementioned kit, wherein the weight ratio of biopolymer tocross-linker is about 30:1 to about 200:1.

In certain embodiments, the present invention relates to theaforementioned kit, wherein the weight ratio of biopolymer tocross-linker is about 60:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned kit, wherein the weight ratio of biopolymer tocross-linker is about 80:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned kit, wherein the weight ratio of biopolymer tocross-linker is about 100:1 to about 120:1.

In certain embodiments, the present invention relates to theaforementioned kit, wherein the weight ratio of biopolymer tocross-linker is about 80:1 to about 100:1.

In certain embodiments, the present invention relates to theaforementioned kit, further comprising a fourth amount of afoam-modifying agent.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said foam-modifying agent is a surfactant.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said foam-modifying agent is tyloxapol, apoloxamer, a poloxamine, a phospholipid, or glycerol.

In certain embodiments, the present invention relates to theaforementioned kit, further comprising a fifth amount of ananti-infective.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said anti-infective is selected from thegroup consisting of an aminoglycoside, a tetracycline, a sulfonamide,p-aminobenzoic acid, a diaminopyrimidine, a quinolone, a β-lactam, aβ-lactamase inhibitor, chloraphenicol, a macrolide, penicillins,cephalosporins, linomycin, clindamycin, spectinomycin, polymyxin B,colistin, vancomycin, bacitracin, isoniazid, rifampin, ethambutol,ethionamide, aminosalicylic acid, cycloserine, capreomycin, a sulfone,clofazimine, thalidomide, a polyene antifungal, flucytosine, imidazole,triazole, griseofulvin, terconazole, butoconazole ciclopirax, ciclopiroxolamine, haloprogin, tolnaftate, naftifine, and terbinafine, or acombination thereof.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said anti-infective is tetracycline.

In certain embodiments, the present invention relates to theaforementioned kit, further comprising a sixth amount of acontrast-enhancing agent.

In certain embodiments, the present invention relates to theaforementioned kit, wherein said contrast-enhancing agent is selectedfrom the group consisting of radiopaque materials, paramagneticmaterials, heavy atoms, transition metals, lanthanides, actinides, dyes,and radionuclide-containing materials.

In certain embodiments, the present invention relates to theaforementioned kit, wherein upon combination of said biopolymer, saidcross-linker and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about10 minutes.

In certain embodiments, the present invention relates to theaforementioned kit, wherein upon combination of said biopolymer, saidcross-linker and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about 7minutes.

In certain embodiments, the present invention relates to theaforementioned kit, wherein upon combination of said biopolymer, saidcross-linker and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 1 minute to about 5minutes.

In certain embodiments, the present invention relates to theaforementioned kit, wherein upon combination of said biopolymer saidcross-linker and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 2 minutes to about6 minutes.

In certain embodiments, the present invention relates to theaforementioned kit, wherein upon combination of said biopolymer saidcross-linker and said polymeric additive, said biopolymer and saidcross-linker are substantially cross-linked in about 3 minutes to about5 minutes.

Therapeutic Applications

In addition to being useful for treating emphysema (e.g., as describedabove and in the following examples), compositions of the invention maybe useful in other therapeutic indications.

Aspects of the invention may be used to treat any form of abnormalcellular growth (e.g., tumors, cancers, etc.) by targeting a non-toxic,yet therapeutically effective, composition to an area of diseased tissue(e.g., a tumor, adenoma, cancer, precancer, or other abnormal lesion).

Another aspect of the invention involves the use of biopolymercompositions to treat solid organ cancer. Examples of such cancersinclude, but are not limited to, bronchogenic carcinoma, malignantmesothelioma. Biopolymer compositions may be used to treat cancers bymethods such as inducing cellular necrosis and/or microvascularthrombosis, which can result in tumor regression.

Another aspect of the invention involves the use of biopolymercompositions to treat pleural effusions. Pleural effusions may be, forinstance, ones that are refractory to medical therapy, such as malignantpleural effusions and benign, but recurrent, pleural effusions.

Another aspect of the invention involves the use of biopolymercompositions to seal bronchopleural fistulas. The compositions can be afoam or a gel. Bronchopleural fistulas may arise from, for example,airway leaks following surgery, lung trauma or invasive infection. Themedical applications of the biopolymer compositions can be applied tothe lung of a patient to seal airway leaks, by filling the airways andalveoli. The biopolymer compositions can be used to permanently seal theinfected site of a patient, by forming a tissue-foam-tissue crosslinkbetween a biopolymer and glutaraldehyde.

In certain embodiments, the present invention relates to a method ofsealing a bronchopleural fistula in a patient, comprising the steps ofadministering a composition comprising a biopolymer, a cross-linker, anda polymeric additive; wherein said polymeric additive accelerates across-linking reaction between the biopolymer and the cross-linker,thereby sealing the fistula.

In certain embodiments, the composition is administered using abronchoscope. In certain embodiments, the composition is administeredusing a catheter. In certain embodiments, the methods of the inventionfurther comprise the step of advancing into a region of a lung of thepatient via the trachea of the patient a catheter lumen through abronchoscope.

Another aspect of the invention involves the use of biopolymercompositions to perform emergency tamponade of bleeding vessels.Examples of bleeding vessels include, but are not limited to, majorinternal limb vessels, gastrointestinal bleeding or internal organbleeding. The biopolymer compositions may be used to treat bleedingvessels following trauma, surgery or gastrointestinal bleeding. Thebiopolymer compositions can be applied to permanently seal a bleedingvessel, by forming a tissue-foam-tissue crosslink between the biopolymerand glutaraldehyde which can act as a localized disinfectant. Thebiopolymer compositions can be applied to post surgical gastrointestinalbleeding thereby sealing the vessel and preventing ongoing blood loss.

Another aspect of the invention involves the use of biopolymercompositions to seal fistulas. Examples of fistulas include, but are notlimited to, fistulas arising from gastrointestinal tumors and postsurgical gastrointestinal fistulas. The biopolymer compositions may beused to seal fistulas in the gastrointestinal tract arising from tumorsor surgery and thereby prevent fluid leakage into the surrounding site.The biopolymer compositions can be applied to permanently seal agastrointestinal fistula, by forming a tissue-foam-tissue crosslinkbetween the biopolymer and glutaraldehyde.

Another aspect of the invention involves the use of biopolymercompositions as a sealant to seal air leaks in a lung after surgery, forexample.

Another aspect of the invention involves a method of attaching a firsttissue to a second tissue of a patient in need thereof comprising,applying to said first tissue or said second tissue or both an effectiveamount of a composition comprising a biopolymer, a cross-linker, and apolymeric additive, thereby attaching said first tissue to said secondtissue.

Another aspect of the invention involves the use of biopolymercompositions as a general topical hemostat. The biopolymer compositionscan be used to control bleeding of, for example, a torn blood vessel.One embodiment of the invention relates to a method of achievinghemostasis, comprising applying to a blood vessel of a patient in needthereof a therapeutically effective amount of a composition comprising abiopolymer, a cross-linker, and a polymeric additive, thereby achievinghemostasis.

Another aspect of the invention involves the use of biopolymercompositions to achieve pleurodesis. The need for pleurodesis may arisefrom refractory medical therapy, such as malignant effusions and pleuralspace diseases. The biopolymer compositions can be used to fill thepleural space and thereby displace the recurrent effusions into thepleural space.

In certain embodiments, the present invention relates to a method ofachieving pleurodesis in a patient, comprising the steps ofadministering a composition comprising a biopolymer, a cross-linker, anda polymeric additive; wherein said polymeric additive accelerates across-linking reaction between the biopolymer and the cross-linker. Incertain embodiments, the composition is administered using a syringe. Incertain embodiments, the composition is administered using a catheter.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example One Bovine Serum Albumin & Glutaraldehyde In Vitro Experiments;Polymerization Time

The following experiments were done to determine the effects of albuminand glutaraldehyde (GA) concentrations on polymerization time. Inaddition, the following experiments were done to determine the effectsof additives (such as polyvinylpyrrolidone and dextran) onpolymerization time of albumin/glutaraldehyde mixtures.

The following reagents and equipment were used: bovine serum albumin(Sigma A7906); polyvinylpyrrolidone (Aldrich 856568); glutaraldehyde 25%w/v (Acros 11998-0010); dextran HMW (Sigma D4876); water; 2 mL microfugetubes; syringes; needles; timer; and a vortex mixer. The following stocksolutions were prepared: a 45% albumin stock in water; a 50% PVP stockin water; and a 50% dextran stock in water.

B. General Procedure

The general procedure used was that a glutaraldehyde mixture (0.1 mL)was added to an albumin mixture (0.9 mL), which may includepolyvinylpyrrolidone or dextran, which was prepared in 2 mL microfugetube. The combination was mixed for 5 seconds with Vortex. The microfugetube inverted every 3 to 15 seconds. Polymerization time recorded whenmixture no longer deforms visibly with inversion.

To measure the effects of albumin and glutaraldehyde (GA) concentrationson polymerization time, serial dilutions of albumin were combined with afixed concentration of GA to assess the effects of albumin concentrationon polymerization time. Albumin at concentrations from 13.5 to 36% waspolymerized with 2% GA.

Serial dilutions of GA were combined with a fixed concentration ofalbumin to assess the effects of GA concentration on polymerizationtime. GA at concentrations from 0.25 to 2% GA was polymerized with 36%albumin.

Albumin and GA were combined at various ratios to assess the effects ofalbumin/GA ratio on polymerization time. Albumin at concentrations from13.5 to 36% was combined with GA at concentrations from 0.25 to 2%,yielding Albumin/GA ratios from 6.75 to 144.

To measure the effects of polyvinylpyrrolidone (PVP) and dextran onpolymerization time, PVP, over a range of concentrations, was added toalbumin mixtures prior to polymerization with GA. For example, 25%albumin with 0 to 15% PVP was polymerized with GA 0.25 to 2%.

In addition, dextran, over a range of concentrations, was added toalbumin mixtures prior to polymerization with GA to assess the effectson polymerization time. For example, 25% albumin with 0 to 15% dextranwas polymerized with GA 0.25 to 2%.

These experiments established that polymerization time varied inverselywith albumin and GA concentrations and albumin/GA ratio. Polymerizationtimes between 1 and 5 minutes could be achieved with albuminconcentrations as low as 13.5% (with 2% GA) and GA concentrations as lowas 0.25% (with 36% albumin). 36% albumin with 2% GA (Bioglue)polymerized in approximately 10 seconds. See FIGS. 1 and 2.

Addition of PVP shortened polymerization time 28.6 to 54.5% in aconcentration dependent manner. Addition of dextran shortenedpolymerization time 15.4 to 33.7% in a concentration dependent manner.See FIGS. 3 and 4.

It can be concluded that albumin and glutaraldehyde can be mixed tocreate a tissue glue with polymerization time suitable foradministration through a thin bore catheter for bronchoscopic lungvolume reduction. In addition, addition of PVP or dextran shortenspolymerization time such that lower concentrations of albumin and/or GAcan be used. This effect may be used to improve the deliverabilityand/or biocompatibility of albumin/GA mixtures with appropriatepolymerization characteristics. Further, the concentrations of albuminand GA in Bioglue® (36% albumin with 2% GA) result in polymerizationtime (approximately 10 seconds) well outside the ideal window forbronchoscopic lung volume reduction (approximately 1 to 5 minutes).

Example Two Human Serum Albumin & Glutaraldehyde In Vitro Experiments;Polymerization Times

The following experiments were done to determine the effect ofsubstituting human serum albumin (HSA) for bovine serum albumin (BSA) onpolymerization time of albumin/glutaraldehyde (GA) mixtures; and todetermine if foams can be generated from such mixtures that can beinjected through a small bore catheter.

The following reagents and equipment were used: 25% Human serum albumin(HSA; Baxter 1500233); polyvinylpyrrolidone (PVP; Aldrich 856568);glutaraldehyde 25% w/v (Acros 11998-0010); 2 mL microfuge tubes;syringes; needles; timer; vortex mixer; three-way stopcocks; and a 5F130 cm single lumen catheter. The following stock solutions wereprepared: 25% HSA with 5% PVP; and 25% HSA with 10% PVP.

In order to measure polymerization times, as described above, an albuminmixture (0.9 mL), which may include polyvinylpyrrolidone or dextran, wasprepared in 2 mL microfuge tube. A glutaraldehyde mixture (0.1 mL) wasthen added. The combination was mixed for 5 seconds with Vortex. Themicrofuge tube inverted every 3 to 15 seconds. Polymerization timerecorded when mixture no longer deforms visibly with inversion.

Serial dilutions of GA were combined with a fixed concentration of HSAto assess the effects of GA concentration on polymerization time. GA atconcentrations from 0.25 to 0.5% GA was polymerized with 22.5% albumin(Note: all concentrations are listed as concentration after finalmixing). The experiments were repeated with the addition of 4.5 or 9%PVP.

In order to measure foamability, an albumin mixture (4.5 mL) was drawninto syringe (30 mL) equipped with three-way stopcock attached. Aglutaraldehyde mixture (0.5 mL) was then attached. Air (5-25 mL) wasthen drawn into second syringe (30 mL) and attached to sideport ofthree-way stopcock. Liquid and air were then mixed by alternatelypushing the plungers of the two syringes (10-20 total pushes) togenerate foam. Foam assessed for stability and homogeneity.

In order to measure injectability, an albumin mixture (4.5 mL) was drawninto syringe (20 mL) equipped with three-way stopcock attached. Aglutaraldehyde mixture (0.5 mL) was then attached. Air (15 mL) was thendrawn into second syringe (20 mL) and attached to sideport of three-waystopcock. Liquid and air were then mixed by alternately pushing theplungers of the two syringes (10-20 total pushes) to generate foam. Theresulting foam was injected through 5F single-lumen catheter by hand.Time to inject and volume of injected foam recorded.

These experiments established that polymerization time varies inverselywith GA concentration. Interestingly, HSA appeared to polymerize morequickly than BSA at similar concentrations. Further, addition of PVPsignificantly shortened polymerization time. The magnitude off theaffect seemed to be larger that seen with BSA under similar conditions.For example, at Liquid:Air ratios of 1:3 or less, 22.5% HSA with 4.5%PVP formed a stable and injectable foam. See FIGS. 5 and 6, as well asTables 1 and 2 (in FIG. 7).

It can therefore be concluded that HSA can be substituted for BSA inalbumin/GA mixtures designed for administration through a thin borecatheter for bronchoscopic lung volume reduction. In addition, additionof PVP has been shown to shorten polymerization time. Finally, 22.5% HSAwith 4.5% PVP and 0.3% GA can be mixed with air at a 1:3 ratio to form afoam stable enough to inject through a small bore catheter.

Example Three Albumin/GA In Vivo Experiments in Sheep

As disclosed in the previous examples, albumin/GA mixtures withdesirable properties for BLVR were identified through a series of invitro experiments. In this example, these formulations were used toperform BLVR in sheep to evaluate efficacy and toxicity of theseformulations.

Anesthesia was induced with ketamine 2 mg/kg, midazolam 0.3 mg/kg, andpropofol 70 mg IV and maintained with propofol continuous infusion.Animals were intubated fiberoptically with a 10 mm oral endotrachealtube and mechanically ventilated with RR 12, TV 500. A baseline CT scanwas obtained at 25 cm H₂O transpulmonary pressure, measured with anesophageal balloon.

The bronchoscope was wedged in a target segmental airway. The deliverycatheter was passed through the working channel of the bronchoscopeuntil its tip was visible 1-2 cm beyond the end of the bronchoscope.

The GA solution was added to the albumin solution and mixed with oxygenfrom a wall source. A foam was generated by pushing the liquid and gasrepeatedly through two syringes connected by a three-way stopcock. Thefoam was drawn into one of the syringes which was attached to theproximal end of the catheter and injected by hand. The whole mixing andinjection procedure took place within approx. 1 minute. The catheter wasthen removed and air was injected through the working channel to pushthe foam distal. After 2-3 minutes, the bronchoscope was removed fromwedge position and the site was inspected for evidence of properpolymerization of the foam.

The bronchoscope was then wedged at the next target segment where theprocedure was repeated. Following completion of the last treatment, arepeat CT scan was obtained at 25 cm H₂O transpulmonary pressure.

In recovery, anesthesia was discontinued and the animal was extubatedand allowed to recover. All sheep were treated with 4 days ofbroad-spectrum antibiotics (Baytril) beginning immediately prior toBLVR. Repeat CT scans were performed at selected timepoints prior toeuthanasia/necropsy. Specifically, six to eight-five days followingBLVR, repeat CT scans were performed at 25 cm H₂O transpulmonarypressure. The animals were then euthanized and necropsied. The abdominaland thoracic organs were inspected. The lungs were removed enbloc andinflated and the treatment sites were evaluated semiquantitatively on ascale of 0 to 3 for their size and contraction. The sites were thendissected and evaluated for evidence of hemorrhage, necrosis, or othergross evidence of toxicity. Tissue samples were taken from each lungtreatment site as well as untreated control sites and preserved in 10%buffered formalin for later histologic processing. Samples of heart,liver, kidney, and spleen were also collected and processed in similarfashion.

In order to measure the effects of albumin and glutaraldehydeconcentrations in vivo, five sheep were treated with five formulationscontaining a range of BSA concentrations from 20 to 38% and GAconcentrations from 0.25 to 0.75%. The animals were followed for 8-9days. See Table 3 (FIG. 7).

CT scans immediately post-BLVR revealed a combination of hazyinfiltrates and denser, more linear infiltrates in all animals. CT scansat one week revealed progression towards denser, more linearinfiltrates. Volume reduction of 22.5 to 76.9 mL per site treated wasdetected by CT integration post treatment. Results at one week were morevariable. Other CT findings at one week included cavities at treatmentsites in sheep 269b, 274, and 256, and pneumomediastinum andpneumothorax in sheep 266. In most cases, the cavities were also visibleon post-treatment CTs.

There was no gross evidence of heart, kidney, liver, or spleen toxicityin any animal. There were no pleural adhesions in any animal. Treatmentsites were easily identified and well localized. Size and contractionscores ranged from 2.13 to 2.88 out a possible 3. The percentage ofsites with hemorrhage/necrosis ranged from 12.5 to 87.5% and was highestin the two groups with the highest GA concentrations. Cavities attreatment sites were identified in animals 269b, 274, 266, and 256.

It can therefore be concluded that the albumin/GA mixtures tested wereeffective in producing bronchoscopic lung volume reduction. In addition,albumin/GA mixtures with higher GA were associated with fevers duringthe first 3 days post treatment. Further, albumin/GA mixtures withhigher GA cause excessive local lung toxicity in the form ofhemorrhage/necrosis. Finally, the albumin/GA mixtures tested sometimesproduce cavities at treatment sites which appear to be immediatemechanical complications of the procedure.

Importantly, attempts to balance polymerization time and toxicity due tohigher glutaraldehyde concentration did not produce a clinicallyworkable solution as on one hand, higher albumin concentration withlower glutaraldehyde led to long polymerization times and to veryviscous solutions which very essentially impossible to inject through anarrow bore catheter, while on the other hand lower albuminconcentrations with higher glutaraldehyde concentrations led to markedtoxicity.

The first set of experiments (described above) indicated that theapproach chosen seemed to induce lung volume reduction; however, toachieve a reasonable operating time, the glutaraldehyde concentrationhad to be relatively high. This was associated with necrosis andhemorrhage at the treatment sites. Therefore, in the next set ofexperiment, the mixture included the polymeric additive, PVP, shown toaccelerate the polymerization of albumin/glutaraldehyde mixtures.

In order to measure the effects of PVP in vivo, eight animals weretreated with a formulation containing 25% BSA, 10% PVP, and 0.25% GA.All animals were treated at a total of eight sites with 4 on each lung).One animal (sheep 143) was treated with 5 mL of the albumin/PVP/GAmixture mixed with 5 mL of oxygen to generate 10 mL of foam. Theremaining animals were treated with 5 mL of the albumin/PVP/GA mixturemixed with 15 mL of oxygen to generate 20 mL of foam. See Table 4 (FIG.8).

It was found that no sheep developed fevers (T>103.5) during the firstthree days following BLVR. Sheep 143 and 4 developed cough increasedrespiratory effort. No animal required additional treatments. Allanimals survived to planned euthanasia/necropsy. CT scans immediatelypost-BLVR revealed a combination of hazy infiltrates and denser, morelinear infiltrates in all animals. CT scans at one week revealedprogression towards denser, more linear infiltrates. Subsequent CT scansshowed persistence of these findings. Volume reduction of 12.6 to 185.9mL per site treated was detected by CT integration at one week. Sheep143 had pneumomediastinum and a pneumothorax as well as a cavity at atreatment site on one week CT scan.

There was no gross evidence of heart, kidney, liver, or spleen toxicityin any animal. There were no pleural adhesions in any animal. Treatmentsites were easily identified and well localized. Size and contractionscores ranged from 1.88 to 2.75 out a possible 3. There was no grosshemorrhage/necrosis in any animal. Sheep 143 had a cavity at onetreatment site.

Therefore it can be concluded that 25% BSA, 10% PVP, 0.25% GA produceseffective bronchoscopic volume reduction. Importantly, there were nofevers and no gross evidence of excessive local pulmonary or systemictoxicity associated with this formulation. Finally, there were fewermechanical complications (cavities at treatment sites) associated withthis formulation compared with previously tested BSA/GA mixtures withoutPVP.

In order to measure the effects of replacing BSA with HSA, four animalswere treated with a formulation containing 22.5% HSA, 4.5% PVP, and 0.3%GA. One animal was treated with 5 mL of the albumin/PVP/GA mixture mixedwith 15 mL of oxygen to generate 20 mL of foam. One animal was treatedwith 10 mL of the albumin/PVP/GA mixture mixed with 20 mL of oxygen togenerate 30 mL of foam. See Table 5 (FIG. 8).

It was found that no sheep developed fevers (T>103.5) during the firstthree days following LVR. Both animals survived to plannedeuthanasia/necropsy. CT scans immediately post-LVR revealed acombination of hazy infiltrates and denser, more linear infiltrates inall animals. CT scans at one week revealed progression towards denser,more linear infiltrates. Volume reduction of 19.6 to 81.2 mL per sitetreated was detected by CT integration at one week. There were no otherCT abnormalities.

Importantly, there was no gross evidence of heart, kidney, liver, orspleen toxicity in any animal. There were no pleural adhesions in anyanimal. Treatment sites were easily identified and well localized. Sizeand contraction scores ranged from 2.25 to 3 out a possible 3. There wasgross evidence of a small amount of hemorrhage/necrosis at one site inanimal 308. There were no cavities at treatment sites in any animal. SeeTable 6 (FIG. 8).

In can therefore be concluded that a composition of 22.5% HSA, 4.5% PVP,0.3% GA produces effect bronchoscopic volume reduction. Remarkably,there were no fevers associated with this treatment. Finally, the smallamount of hemorrhage/necrosis seen at one treatment site in one sheepdid not appear to be clinically significant.

Example Four Long-Term Lung Volume Reduction Treatment in Healthy Sheep

The following formulation was tested in-vivo in 4 healthy, female sheep:25% BSA, 6% PVP, 0.25% GA

TABLE 1 Treatment groups Days from treatment to Sheep Group Sitesnecropsy 652 BSA/PVP 6 92 659 6 92 602 6 90 605 6 90

Anesthesia was induced with ketamine 2 mg/kg, midazolam 0.3 mg/kg, andpropofol 70 mg IV and maintained with propofol continuous infusion.Animals were intubated fiberoptically with a 10 mm oral endotrachealtube and mechanically ventilated with RR 12, TV 500. A baseline CT scanwas obtained at 25 cm H₂O transpulmonary pressure, measured with anesophageal balloon.

Simultaneous recordings of flow and transpulmonary pressure werecollected during tidal breathing using a pneumotach and pressuretransducer and WinDaq data acquisition system. The same setup was usedto collect pressure and volume data during stepwise deflation fromtranspulmonary pressure≧30 to FRC.

The bronchoscope was wedged in a target segmental airway. The deliverycatheter was passed through the working channel of the bronchoscopeuntil its tip was visible 1-2 cm beyond the end of the bronchoscope. TheGA solution was added to the Albumin/PVP solution and mixed with oxygenfrom a wall source. Foam was generated by pushing the liquid and gasrepeatedly through two syringes connected by a three-way stopcock. Thefoam was drawn into one of the syringes, which was attached to theproximal end of the catheter and injected by hand. The catheter was thenremoved and air was injected through the working channel to push thefoam distal. After 2-3 minutes, the bronchoscope was removed from wedgeposition and the site was inspected for evidence of properpolymerization of the foam. The bronchoscope was then wedged at the nexttarget segment where the procedure was repeated. Five mL of liquid wascombined with 15 mL of 100% O₂ yielding 20 mL of foam at each treatmentsite. 6 sites were treated in the caudal and middle lobes of each sheep,depending on the size of the sheep. Sites were distributed betweendorsal and lateral sites. Following completion of the last treatment, arepeat CT scan was obtained at 25 cm H₂O transpulmonary pressure.Physiology measurements were also repeated.

In recovery, anesthesia was discontinued and the animal was extubatedand allowed to recover. All sheep were treated with 4 days ofbroad-spectrum antibiotics (Baytril) beginning immediately prior totreatment. Repeat CT scans and physiology were performed at 1, 4, 8, and12 weeks. At 12 weeks following treatment, repeat CT scans wereperformed at 25 cm H₂O transpulmonary pressure. The animals were theneuthanized and necropsied. The abdominal and thoracic organs wereinspected. The lungs were removed enbloc and inflated and the treatmentsites were evaluated semiquantitatively on a scale of 0 to 3 for theirsize and contraction. The sites were then dissected and evaluated forevidence of hemorrhage, necrosis, or other gross evidence of toxicity.Tissue samples were taken from each lung treatment site as well asuntreated control sites and preserved in 10% buffered formalin for laterhistologic processing. Samples of heart, liver, kidney, and spleen werealso collected and processed in similar fashion.

Based on our clinical findings, the sheep used in the experimentgenerally tolerated the lung volume reduction procedure well. CT scanswere analyzed quantitatively using the Emphylxj software. Changes intotal volume, right side (treated) volume, and right side normalized toleft side (to account for differences in inflation pressure) weredivided by the number of sites treated to yield volume changes per site.The results are shown in FIG. 9. Flow data were integrated to yieldvolume and corrected for drift. Resistance(R) and compliance(C) werecalculated by the covariance method using a Matlab program. Pressurevolume curves were generated from the step-wise deflation data and fitto the equation of Salazar and Knowles to yield Vmax, A, k, and Vminusing a separate Matlab program. There were no physiological changes inVmax and A between baseline and 12 weeks.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications citedherein are hereby incorporated by reference.

EQUIVALENTS

While several embodiments of the present invention are described andillustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

1-26. (canceled)
 27. A method of sealing a bronchopleural fistula in apatient, comprising the steps of administering to a patient in needthereof a therapeutically effective amount of a composition comprising abiopolymer, a cross-linker, and a polymeric additive; wherein saidpolymeric additive accelerates a cross-linking reaction between thebiopolymer and the cross-linker, thereby sealing the fistula.
 28. Amethod of achieving pleurodesis in a patient, comprising the steps ofadministering to a patient in need thereof a therapeutically effectiveamount of a composition comprising a biopolymer, a cross-linker, and apolymeric additive; wherein said polymeric additive accelerates across-linking reaction between the biopolymer and the cross-linker. 29.A method of sealing an air leak in a lung, comprising administering to alung of a patient in need thereof a therapeutically effective amount ofa composition comprising a biopolymer, a and cross-linker, and apolymeric additive, thereby sealing the air leak in the lung.
 30. Amethod of attaching a first tissue to a second tissue of a patient inneed thereof comprising, applying to said first tissue or said secondtissue or both an effective amount of a composition comprising abiopolymer, a cross-linker, and a polymeric additive, thereby attachingsaid first tissue to said second tissue.
 31. A method of achievinghemostasis, comprising applying to a blood vessel of a patient in needthereof a therapeutically effective amount of a composition comprising abiopolymer, a cross-linker, and a polymeric additive, thereby achievinghemostasis.
 32. A method of administering emergency tamponade to ableeding vessel in a patient, comprising: administering to a bleedingvessel of a patient a therapeutically effective amount of a compositioncomprising a biopolymer, a and cross-linker, and a polymeric additive,thereby sealing the vessel.
 33. A method of administering emergencytamponade to a gastrointestinal vessel in a patient, comprising:administering to a gastrointestinal vessel of a patient atherapeutically effective amount of a composition comprising abiopolymer, a and cross-linker, and a polymeric additive, therebysealing the vessel.
 34. A method of administering emergency tamponade toan internal organ in a patient, comprising: administering to an internalorgan of a patient in need thereof a therapeutically effective amount ofa composition comprising a biopolymer, a cross-linker, and a polymericadditive, thereby preventing the organ from bleeding.